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Clinical features of culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia OBJECTIVE: This retrospective chart review describes the epidemiology and clinical features of 40 patients with culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. METHODS: Patients with positive M. pneumoniae cultures from respiratory specimens from January 1997 through December 1998 were identified through the Microbiology records. Charts of patients were reviewed. RESULTS: 40 patients were identified, 33 (82.5%) of whom required admission. Most infections (92.5%) were community-acquired. The infection affected all age groups but was most common in infants (32.5%) and pre-school children (22.5%). It occurred year-round but was most common in the fall (35%) and spring (30%). More than three-quarters of patients (77.5%) had comorbidities. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common symptoms, and crepitations (60%), and wheezes (40%) were the most common signs. Most patients with pneumonia had crepitations (79.2%) but only 25% had bronchial breathing. Immunocompromised patients were more likely than non-immunocompromised patients to present with pneumonia (8/9 versus 16/31, P = 0.05). Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications, 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities. CONCLUSION: our results were similar to published data except for the finding that infections were more common in infants and preschool children and that the mortality rate of pneumonia in patients with comorbidities was high. Mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections. It remains one of the most frequent causes of atypical pneumonia particu-larly among young adults. [1, 2, 3, 4, 5] Although it is highly transmissible, most infections caused by this organism are relatively minor and include pharyngitis, tracheobronchitis, bronchiolitis, and croup with one fifth of in-fections being asymptomatic. [6, 7] Only 3 -10% of infected subjects develop symptoms consistent with bronchopneumonia and mortality from infection is rare. [6, 7] The organism is fastidious and difficult to grow on cultures. Therefore, diagnosis of infections caused by this organism is usually confirmed with serological tests or polymerase chain reaction-gene amplification techniques. At King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, the facility to perform Mycoplasma culture has been available since January 1997. As published information concerning M. pneumoniae infections in Saudi Arabia is scarce, [8, 9, 10] we wished to study the epidemiology and clinical features of cultureproven infections caused by this organism at this hospital.

KAUH is a tertiary care teaching hospital with a bed capacity of 265 beds and annual admissions of 18000 to 19000 patients. Patients with M. pneumoniae positive cultures from respiratory specimens were identified over a 24-months" period from January, 1997 through December, 1998 for this review.

During the study period, respiratory specimens (sputum, nasopharyngeal aspiration, endotracheal secretion, and bronchoalveolar lavage) for M. pneumoniae culture were obtained from patients with upper or lower respiratory tract infections seen as inpatients or in the outpatient or emergency departments. Respiratory specimens were aslo Gram-stained and cultured for bacteria and viruses. M. pneumoniae serological tests for IgG or IgM were not available at KAUH during the study period. All positive culture results were obtained from the Microbiology laboratory records. Charts of patients were reviewed with standardized data collection. Information collected included patients' demographics, comorbidities, clinical manifestations, complications, and outcome.

M. pneumoniae was cultured using the classic M. pneumoniae agar medium (M.P. agar) and the Pneumofast tray (Pneumofast ® , International Microbio, Signes, France). Specimens were processed according to the instructions of the manufacturer. The M.P. agars and Pneumofast trays were incubated anaerobically at 37°C and inspected daily for 4 weeks. The organism was identified based on typical colonial morphology (granular colonies, rarely fried-egg-like, 10-150 ∝ in diameter) on the M.P. agar medium and the change in the Pneumofast broth color from red to orange then to yellow (glucose fermentation) in the absence of turbidity of the broth. Antibiotic sensitivity profile on the Pneumofast tray was also used for identification according to the instructions of the manufacturer. Bacterial and viral cultures were performed using standard methods.

M. pneumoniae isolates were considered community-acquired if they were recovered from unhospitalized patients or within 72 hours of admission to the hospital, and nosocomial if they were recovered beyond that period.

Pneumonia was diagnosed based on clinical symptoms and signs, along with radiographic evidence of pneumonia when possible. Severe pneumonia was defined as pneumonia associated with tachycardia (>140 /minute), tachypnoea (>30/minute), hypotension (Systolic blood pressure <90 mmHg), hypoxemia (arterial oxygen partial pressure <8 kPa or oxygen saturation <90%), and/or more than 2 areas of consolidation.

Outcome of patients with M. pneumoniae infection was classified into 4 categories; uneventful recovery, recovery following complications, death due to M. pneumoniae infection, or death unrelated to M. pneumoniae infection.

The Statistical Package for Social Sciences (SPSS) program was used for data analysis. Comparison of categorical data was by Chi-square statistic or Fisher's exact test for small expected values.

A total of 40 respiratory specimens from 40 patients were positive for M. pneumoniae over the 24-months study period. The demographic and epidemiological characteristics of the patients are summarized in Table 1 . Of all isolates, 37 (92.5%) were community-acquired and 3 (7.5%) were nosocomial. Thirty-three (82.5%) patients required admission to the hospital and the remaining 7 (17.5%) were treated as outpatients. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Of the 24 cases of pneumonia, 21 were confirmed radiologically and the remaining 3 were diagnosed clinically. The two cases of bronchiolitis occurred in 2 children, one and three years old. Thirty-one patients (77.5%) had comorbidities. Eleven patients (27.5%) had cardiopulmonary comorbidities (asthma, 8, lung fibrosis, 1, congestive heart failure, 1, congenial heart disease, 1), 9 patients (22.5%) were immunocompromised (malignancy, 7, steroid therapy, 3, Human immunodeficiency virus infection, 1), and 11 patients (27.5%) had other comorbidities (premature newborns, 2, and one each of myelodysplastic syndrome, myelopro-liferative disorder, sickle cell anemia, Evan's syndrome, Down syndrome, sarcoidosis, demyelinating disease, cerebral palsy, and spinal muscle atrophy). Organisms concomitantly isolated with M. pneumoniae from the respiratory tract included herpes simplex virus type 1 (2 occasions), adenovirus (2 occasions), cytomegalo virus (1 occasion), respiratory syncytial virus (1 occasion), and bacterial isolates (2 occasions: Acinetobacter species, 1, and Enter obacter cloacae, 1).

Clinical manifestations associated with M. pneumoniae infections are summarized in Table 2 . Pneumonia was more common than upper respiratory tract infections (57.5 % versus 27.5%, respectively). Immunocompromised patients were more likely to present with pneumonia as opposed to upper respiratory tract infection or bronchiolitis than non-immunocompromised patients (8/9 versus 16/31, P = 0.05). Similarly, there was a tendency for patients 60 years of age or older to present with pneumonia more frequently than those below 60 (4/4 versus 20/36, P = 0.1). Of the 24 patients with clinically or radiologically confirmed pneumonia, 19 (79.2%) had crepitations and only 6 (25%) had bronchial breath sounds on physical examination. Of the 16 patients in whom wheezes were detected, 9 (56.3%) were not known to have asthma or other obstructive airway disease. Table 3 . Of the 24 patients with pneumonia, 21 (87.5%) were admitted to the hospital, and 20 (83.3%) had comorbidities. All patients with upper respiratory tract infections (11 patients) or bronchiolitis (2 patients) had uneventful recovery. Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications (acute respiratory distress syndrome, 2, respiratory failure, 1, septic shock, 1), 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities; one had congestive heart failure, the second had congenital heart disease, and the third was a 3months old infant born prematurely at 32 weeks of gestation who previously had 3 episodes of pneumonia due to other pathogens.

Mycoplasma pneumoniae is one of the most common causes of atypical pneumonia accounting for 5-23% of community-acquired pneumonia, [1, 2, 3, 4, 5] In a study of 511 children with acute respiratory tract infection in Riyadh, Saudi Arabia, Mycoplasma pneumoniae was found to be the second most common causative agent after Respiratory syncytial virus (RSV) accounting for 9% of all cases, [8] In a study of 112 adult patients with community acquired pneumonia admitted to a military hospital in Riyadh, Saudi Arabia, this organism accounted for 6% of all cases, [9] In another retrospective study of 567 pneumonic episodes in adult patients from Al-Qassim area, the organism accounted for 23% of all episodes, [10] The organism also causes other relatively minor infections such as pharyngitis, tracheobronchitis, bronchiolitis, and croup. It is transmitted from person-to-person by infected respiratory droplets during close contact. It is most common in school-aged children, military recruits, and college students. [11] Most cases occur singly or as family outbreaks. Larger outbreaks can also occur in closed populations such as military recruit camps or boarding schools, [12] Infection occurs most frequently during the fall and winter in temperate climates but may develop year-round, [13] The average incubation period is 3 weeks following exposure, [6] Although rare, complications are protean and may involve virtually any organ system such as the respiratory system (e.g.: pleurisy, pneumothorax, acute respiratory distress syndrome, lung abscess), the hematologic system (e.g.: hemolytic anemia, intravascular coagulation, thrombocytopenia), the dermatologic system (e.g.: maculopapular or urticarial rashes, erythema multiforme, erythema nodosum), the musculoskeletal system (e.g.: myalgias, arthralgias, arthritis), the cardiovascular system (e.g.: pericarditis, myocarditis), the nervous system (e.g.: meningoencephalitis, Guillain-Barre syndrome, neuropathies, acute psychosis), or the eye (optic disc edema, optic nerve atrophy, retinal exudation and hemorrhages). [6, 7, 14, 15, 16, 17, 18] Immunity following infection is not long lasting. [11] In our study, the infection affected all age groups but was most common in infants (32.5%) and preschool children (22.5%), and least common in adults aged 15 to 30 years (2.5%) and elderly above 70 years of age (5%). This contrasts with data from temperate countries where the infection is most common in school-aged children, and young adults. [11] One possible explanation for this difference is that infants and preschool children perhaps had more severe infections than did school-aged children, and young adults which prompted presentation of the former group to the hospital. The infection occurred year-round but was most common in the fall (35%), and spring (30%), and least common in the summer (10%). Most infections were community-acquired (92.5%).

More than one half of patients (57.5%) presented with pneumonia, and about a third (27.5%) presented with upper respiratory tract infection, Immunocompromised patients and patients 60 years of age or older were more likely to present with pneumonia as opposed to upper respiratory tract infection than non-immunocompromised patients or those below 60 years of age. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common presenting symptoms. Cough was usually dry or slightly productive of white sputum and mild to moderate in severity. Most febrile patients had mild to mod- erate fever of 39°C or less; high-grade fever of more than 39°C was rare. Crepitations (60%), and wheezes (40%) were the most common signs. Wheezes were as common in patients with no history of obstructive airway disease (9 patients) as it was in those with such a history (7 patients). Bronchial breathing as a sign of consolidation was detected in only one fourth of patients with pneumonia, which is consistent with the known disparity between clinical and radiological signs of M pneumoniae pneumonia. Crepitations, however, were detected in the majority (79.2%) of patients. Pleuritic chest pain and pleural effusion were rare.

More than half (56.5%) of the patients with pneumonia had uneventful recovery. Mortality from M. pneumoniae pneumonia was high (12.5%) and occurred only in patients with underlying comorbidities. None of the 9 patients with no underlying comorbidities died of M pneumoniae pneumonia. The relatively high complications rate (16.7%) and mortality (12.5%) related to M. pneumoniae pneumonia are likely due to selection bias as most patients with pneumonia were sick enough to require admission to the hospital (21/24 or 87.5%) and most of them had comorbidities (20/24 or 83.3%).

In conclusion, our data shed some light on the epidemiology and clinical features of M pneumoniae infections in one of the Saudi tertiary care centers. The data are comparable to those of other countries except for the finding that infections were more common in infants and preschool children than in school children and young adults. Additionally, mortality attributable to M. pneumoniae pneumonia was relatively high in patients with comorbidities. It is hoped this information will assist clinicians in their approach and management of respiratory tract infections. Nitric oxide: a pro-inflammatory mediator in lung disease? Inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO•) and increased indices of NO• -dependent oxidative stress. Although NO• is known to have anti-microbial, anti-inflammatory and anti-oxidant properties, various lines of evidence support the contribution of NO• to lung injury in several disease models. On the basis of biochemical evidence, it is often presumed that such NO• -dependent oxidations are due to the formation of the oxidant peroxynitrite, although alternative mechanisms involving the phagocyte-derived heme proteins myeloperoxidase and eosinophil peroxidase might be operative during conditions of inflammation. Because of the overwhelming literature on NO• generation and activities in the respiratory tract, it would be beyond the scope of this commentary to review this area comprehensively. Instead, it focuses on recent evidence and concepts of the presumed contribution of NO• to inflammatory diseases of the lung. Since its discovery as a biological messenger molecule more than 10 years ago, the gaseous molecule nitric oxide (NO • ) is now well recognized for its involvement in diverse biological processes, including vasodilation, bronchodilation, neurotransmission, tumor surveillance, antimicrobial defense and regulation of inflammatory-immune processes [1] [2] [3] . In the respiratory tract, NO • is generated enzymically by three distinct isoforms of NO • synthase (NOS-1, NOS-2 and NOS-3) that are present to different extents in numerous cell types, including airway and alveolar epithelial cells, neuronal cells, macrophages, neutrophils, mast cells, and endothelial and smoothmuscle cells. In contrast with the other two NOS isoforms (NOS-1 and NOS-3), which are expressed constitutively and activated by mediator-induced or stress-induced cell activation, NOS-2 activity is primarily regulated transcriptionally and is commonly induced by bacterial products and pro-inflammatory cytokines. As such, inflammatory diseases of the respiratory tract, such as asthma, acute respiratory distress syndrome (ARDS) and bronchiectasis, are commonly characterized by an increased expression of NOS-2 within respiratory epithelial and inflammatory-immune cells, and a markedly elevated local production of NO • , presumably as an additional host defense mechanism against bacterial or viral infections. The drawback of such excessive NO • production is its accelerated metabolism to a family of potentially harmful reactive nitrogen species (RNS), including peroxynitrite (ONOO -) and nitrogen dioxide (NO 2 • ), especially in the presence of phagocyte-generated oxidants. The formation of such RNS is thought to be the prime reason why NO • can in many cases contribute to the etiology of inflammatory lung disease [4] [5] [6] . Despite extensive research into both pro-inflammatory and anti-inflammatory actions of NO • , the overall contribution of NO • to inflammatory conditions of the lung is not easily predicted and seems to depend on many factors, such as the site, time and degree of NO • production in relation to the local redox status, and the acute or chronic nature of the immune response. In addition, our current understanding of the pro-inflammatory or pro-injurious mechanisms of NO • or related RNS is incomplete; this commentary will focus primarily on these latter aspects.

To explore a role for NO • (or NOS) in infectious or inflammatory diseases, two general research approaches have been taken: the use of pharmacological inhibitors of NOS isoenzymes, and the targeted deletion of individual NOS enzymes in mice. Both approaches suffer from the shortcoming that animal models of respiratory tract diseases generally do not faithfully reflect human disease. The use of NOS inhibitors to determine the contribution of individual NOS isoenzymes is also hindered by problems related to specificity and pharmacokinetic concerns. However, the unconditional gene disruption of one or more NOS isoforms, leading to lifelong deficiency, can have a markedly different outcome from pharmacological inhibition at a certain stage of disease, as the involvement of individual NOS isoenzymes can be different depending on disease stage and severity. Despite these inherent limitations, studies with the targeted deletion of NOS isoforms have led to some insights, indicating a role for NO • and NOS-2 in the etiology of some inflammatory lung diseases. For instance, mice deficient in NOS-2 are less susceptible to lethality after intranasal inoculation with influenza A virus, suffer less lung injury after administration of endotoxin, and display reduced allergic eosinophilia in airways and lung injury in a model of asthma, than their wild-type counterparts [7] [8] [9] . However, although the contribution of NOS-2 is expected in inflammatory conditions, recent studies have determined that NOS-1, rather than NOS-2, seems to be primarily involved in the development of airway hyper-reactivity in a similar asthma model [10] . The linkage of NOS-1 to the etiology of asthma was more recently supported in asthmatic humans by an association of a NOS-1 gene polymorphism with this disease, although the physiological basis for this association remains unclear [11] .

Despite the potential contribution of NOS-2-derived NO • to lung injury after endotoxemia, the sequestration of neutrophils in the lung and their adhesion to postcapillary and postsinusoidal venules after administration of endotoxin were found to be markedly increased in NOS-2-deficient mice, and NOS-2 deficiency did not alleviate endotoxininduced mortality. It therefore seems that the 'harmful' and 'protective' effects of NOS-2 might contend with each other within the same model, which makes the assessment of the potential role of NOS in human disease even more difficult. In this context, it is interesting to note that humans or animals with cystic fibrosis have subnormal levels of NOS-2 in their respiratory epithelium, related to a gene mutation in the cystic fibrosis transmembrane conductance regulator [12] . This relative absence of epithelial NOS-2 might be one of the contributing factors behind the excessively exuberant respiratory tract inflammatory response in patients with cystic fibrosis, even in the absence of detectable respiratory infections. Overall, the apparently contrasting findings associated with NOS deficiency, together with concerns about animal disease models used, make interpretations and conclusions with regard to human lung disease all the more difficult.

Pharmacological inhibitors of NOS have also been found to reduce oxidative injury in several animal models of lung injury, such as ischemia/reperfusion, radiation, paraquat toxicity, and endotoxemia (see, for example, [13] [14] [15] ). However, results are again not always consistent, and in some cases NOS inhibition has been found to worsen lung injury, indicating anti-inflammatory or protective roles for NO • . All in all, despite these inconsistencies, there is ample evidence from such studies to suggest a contributing role of NO • in various respiratory disease conditions, which continues to stimulate research into mechanistic aspects underlying such pro-inflammatory roles and modulation of NO • generation as a potential therapeutic target.

Although the pro-inflammatory and injurious effects of NO • might be mediated by a number of diverse mechanisms, it is commonly assumed that such actions are largely due to the generation of reactive by-products generated during the oxidative metabolism of NO • ; these are collectively termed RNS. One of the prime suspects commonly implicated in the adverse or injurious properties of NO • is ONOO -, a potent oxidative species formed by its almost diffusion-limited reaction with superoxide (O 2

•-), which is a product of activated phagocytes and of endothelial or epithelial cells [4, 5, 13] . The formation of ONOOseems highly feasible under conditions of elevated production of both NO • and O 2

•in vivo, and its oxidative and cytotoxic potential is well documented [5, 6] . However, because the direct detection of ONOOunder inflammatory conditions is virtually impossible because of its instability and high reactivity, the formation of ONOOin vivo can be demonstrated only by indirect methods. Thus, many investigators have relied on the analysis of characteristic oxidation products in biological molecules, such as proteins and DNA, most notably free or protein-associated 3-nitrotyrosine, a product of tyrosine oxidation that can be formed by ONOO -(and several other RNS) but not by NO • itself (see, for commentary review reports primary research http://respiratory-research.com/content/1/2/067 example, [5] ). Indeed, elevated levels of 3-nitrotyrosine have been observed in many different inflammatory conditions of the respiratory tract [16] , which illustrates the endogenous formation of ONOOor related RNS in these cases. However, without known evidence for functional consequences of (protein) tyrosine nitration, the detection of 3-nitrotyrosine should not be regarded as direct proof of a pro-inflammatory role of NO • . Moreover, although the detection of 3-nitrotyrosine has in most cases been interpreted as conclusive evidence for the formation of ONOOin vivo (see, for example, [17] ), it should be realized that other RNS formed by alternative mechanisms might also contribute to endogenous tyrosine nitration. Indeed, it has recently become clear that the presence of inflammatory-immune cells, and specifically their heme peroxidases myeloperoxidase (MPO) and eosinophil peroxidase (EPO), can catalyze the oxidization of NO • and/or its metabolite NO 2 to more reactive RNS and thereby contribute to protein nitration [16, 18, 19] . This notion is further supported by the fact that 3-nitrotyrosine is commonly detected in tissues affected by active inflammation, mostly in and around these phagocytic cells and macrophages, which can also contain active peroxidases originating from apoptotic neutrophils or eosinophils. Hence, the detection of 3-nitrotyrosine in vivo cannot be used as direct proof of the formation of ONOO -, but merely indicates the formation of RNS by multiple oxidative pathways, possibly including ONOObut more probably involving the activity of phagocyte peroxidases [16, 20] . In this regard, a preliminary study with EPO-deficient mice has recently demonstrated the critical importance of EPO in the formation of 3-nitrotyrosine in a mouse model of asthma [21] . Future studies with animals deficient in MPO and/or EPO will undoubtedly help to clarify this issue.

Given the considerable interest in 3-nitrotyrosine as a collective marker of the endogenous formation of NO •derived RNS, the crucial question remains of whether the detection of 3-nitrotyrosine adequately reflects the toxic or injurious properties of NO • . The formation of ONOO -(or of other RNS that can induce tyrosine nitration) might in fact represent a mechanism of decreasing excessive levels of NO • that might exert pro-inflammatory actions by other mechanisms. For instance, NO • can promote the expression of pro-inflammatory cytokines or cyclo-oxygenase (responsible for the formation of inflammatory prostanoids) by mechanisms independent of ONOO - [22, 23] , and the removal of NO • would minimize these responses. Furthermore, although ONOOor related NO •derived oxidants can be cytotoxic or induce apoptosis, these effects might not necessarily relate to their ability to cause protein nitration (see, for example, [16]). For instance, the bactericidal and cytotoxic properties of ONOOare minimized by the presence of CO 2 , even though aromatic nitration and other radical-induced modifications are enhanced [5] . Similarly, the presence of NO 2 in the incubation medium decreases the cytotoxicity of MPO-derived hypochlorous acid (HOCl) toward epithelial cells or bacteria, despite increased tyrosine nitration of cellular proteins (A van der Vliet and M Syvanen, unpublished data). Thus, it would seem that the cytotoxic properties of NO • and/or its metabolites might instead be mediated through preferred reactions with other biological targets, and these might not necessarily be correlated with the degree of tyrosine nitration. The extent of nitrotyrosine immunoreactivity in bronchial biopsies of asthmatic patients was correlated directly with measured levels of exhaled NO • and inversely with the provocation concentration for methacholine (PC 20 ) and forced expiratory volume in 1 s [24] . However, an immunohistochemical analysis of nitrotyrosine and apoptosis in pulmonary tissue samples from lung transplant recipients did not identify patients with an imminent risk of developing obliterative bronchiolitis [25] . It is therefore still unclear to what degree tyrosine nitration relates to disease progression.

Several studies with purified enzymes have suggested that nitration of critical tyrosine residues adversely affects enzyme activity, but there is as yet no conclusive evidence in vivo for biological or cellular changes as a direct result of tyrosine nitration [16, 20] . For instance, tyrosine nitration was suggested to have an effect on cellular pathways by affecting cytoskeletal proteins or tyrosine phosphorylation, thereby affecting processes involved in, for example, cell proliferation or differentiation [16, 26] . Recent studies have provided support for selective tyrosine nitration within certain proteins [27, 28] and of selective cellular targets for nitration by RNS (see, for example, [29, 30] ), and such specificity might indicate a potential physiological role for this protein modification. However, in none of these cases could tyrosine nitration be linked directly to changes in enzyme function. Chemical studies have indicated that tyrosine nitration by RNS accounts for only a minor fraction of oxidant involved, and reactions with other biological targets (thiols, selenoproteins, or transition metal ions) are much more prominent [5, 6] . Indeed, the extent of tyrosine nitration in vivo is very low (1-1000 per 10 6 tyrosine residues according to best estimates [16]), although different analytical methods used to detect 3-nitrotyrosine in biological systems have often given inconsistent results. It is important to note that recent rigorous studies have unveiled substantial sources of artifact during sample preparation, which might frequently have led to an overestimation of tyrosine nitration in vivo in previous studies [31] .

On the basis of current knowledge, the formation of 3-nitrotyrosine seems to be merely a marker of NO •derived oxidants, with as yet questionable pathophysiological significance. In view of the low efficiency of tyrosine nitration by biological RNS, and the endogenous presence of variable factors that influence protein nitration (antioxidants or other RNS scavengers), it seems unlikely that tyrosine nitration is a reliable mechanism of, for example, enzyme regulation. Nevertheless, the recent discovery of enzymic 'denitration' mechanisms that can reverse tyrosine nitration [32] merits further investigation of the possibility that tyrosine nitration might reflect a signaling pathway, for example analogous to tyrosine phosphorylation or sulfation.

The biological effects of NO • are mediated by various actions, either by NO • itself or by secondary RNS, and the overall biochemistry of NO • is deceptively complex. Moreover, the metabolism and chemistry of NO • depend importantly on local concentrations and pH; the recently described acidification of the airway surface in asthmatics [33] might significantly affect NO • metabolism in these patients. It is well known that interactions with the ion centers of iron or other transition metals are responsible for many of the signaling properties of NO • ; the activation of the heme enzyme guanylyl cyclase and the consequent formation of cGMP is involved not only in smooth-muscle relaxation but also in the activation of certain transcription factors, the expression of several pro-inflammatory and anti-inflammatory genes (including cytokines and cyclo-oxygenase), and the production of respiratory mucus [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . In addition to such direct signaling properties, many actions of NO • might be due largely to secondary RNS that can react with multiple additional targets, in some cases forming nitroso or nitro adducts as potentially unique NO • -mediated signaling mechanisms. As discussed, the formation of protein nitrotyrosine has been postulated as a potential RNSspecific signaling pathway. Even more interest has been given to the reversible S-nitros(yl)ation of protein cysteine residues, which has been proposed to affect a number of redox-sensitive signaling pathways, for example by the activation of p21 ras or the inhibition of protein tyrosine phosphatases [35, 36] . Similar modifications of reactive cysteine residues in transcription factors such as nuclear factor-κB or of caspases contribute to the regulation of gene expression and apoptosis [37] [38] [39] . The precise mechanisms leading to protein S-nitrosylation in vivo are still not clarified, but might involve dinitrogen trioxide (formed during the autoxidation of NO • ), iron-nitrosyl complexes, and perhaps ONOO - [16] ; changes in NO • metabolism during inflammatory lung diseases undoubtedly affect such NO • -dependent signaling pathways. In addition, S-nitrosylation can be reversed by either enzymic (thioredoxin or glutaredoxin) or chemical (metals or oxidants) mechanisms, and evidence is increasing that this reversible modification is complementary to more widely accepted oxidant-dependent redox signaling pathways [40] . The reported alterations in S-nitrosothiol levels in tracheal secretions of patients with asthma or cystic fibrosis further point to altered NO • metabolism in these cases, and might provide new clues to the role of S-nitrosylation in controlling such disease processes [41, 42] . Unfortunately, technical limitations to detect S-nitrosylation in specific protein targets in vivo have limited a full understanding of this potential signaling pathway; further research in these areas can be expected to establish more clearly its significance in the pathophysiological properties of NO • .

Despite the by now overwhelming evidence for the increased formation of NO • and NO • -derived oxidants in many different lung diseases, the exact contribution of NO • or its metabolites to inflammatory lung disease is still unclear. Indeed, NO • might have distinctly different roles in different stages of respiratory tract inflammatory diseases, being pro-inflammatory or pro-injurious in acute and severe stages but perhaps being protective and antiinflammatory in more stable conditions; it is uncertain whether NOS is a suitable therapeutic target in the management of inflammatory lung disease. Caution is clearly needed when interpreting observations of tyrosine nitration in animal models of disease or in human tissues, which does not automatically implicate ONOO -(as often thought), but rather indicates the formation of RNS by various mechanisms. Furthermore, animal models of chronic lung disease that usually reflect short-term or acute inflammation might not always be applicable to chronic airway diseases in humans. For instance, phagocyte degranulation, a common feature observed in association with human airway inflammatory diseases such as asthma, does not seem to occur in mouse models of asthma [43] . Therefore the importance of granule proteins, such as heme peroxidases, in the pathology of human airway diseases might not be adequately reflected in such animal models. More work with animal models more characteristic of human diseases or with biopsy materials from human subjects will be required to unravel the precise role of NO • in inflammatory lung disease, and might establish more clearly whether the pharmacological inhibition of NOS isoenzymes can be beneficial. This brings up the interesting paradox that, despite presumed adverse roles of NO • in such inflammatory lung diseases as septic shock and ARDS, NO • inhalation has been suggested as a potential therapeutic strategy to improve overall gas exchange [44] . Intriguingly, in a rat model of endotoxemia, inhalation of NO • was found to reduce neutrophilic inflammation and protein nitration [45] , again supporting the crucial involvement of inflammatory-immune cells in this protein modification.

For a better assessment of the role of NO • in respiratory tract diseases in humans, the production of RNS and/or characteristic markers would need to be more carefully monitored during various disease stages. Care should be given to analytical techniques, their quantitative capacity and the possibility of artifacts. The monitoring of exhaled NO • , although convenient and non-invasive, does not reflect the actual production or fate of NO • in the respiratory tract and is not well correlated with NOS activity in the lung [46] . We therefore need to continue research into the local biochemistry of NO • in the lung, taking into account the presence of secreted or phagocyte peroxidases and possible changes in local pH, as in asthmatic airways [33] , that might modulate NO • activity and metabolism. This might result in a better understanding of relationships between the various metabolic endproducts of NO • (NO 2 -, NO 3 -, or nitroso and nitro adducts) and its pro-inflammatory or injurious properties. Surfactant protein-D and pulmonary host defense Surfactant protein-D (SP-D) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. SP-D is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. SP-D, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. SP-D also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. In addition, SP-D binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. Consistent with these diverse in vitro activities is the observation that SP-D-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. The phenotype of macrophages isolated from the lungs of SP-D-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. The expression of SP-D is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized SP-D might contribute to the pathogenesis of a variety of human lung diseases. Surfactant protein-D (SP-D) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein A (SP-A) and the serum mannose-binding lectin [1] [2] [3] . Collectins inter-act with a wide variety of microorganisms, lipids, and organic particulate antigens, and can modulate the function of immune effector cells and their responses to these ligands. This article reviews what is currently known about the sites of production, structure, function, and regulated expression of SP-D. Emphasis will be placed on functional attributes, known ligand interactions, and structure-function relationships believed to be important for host defense. For additional information on SP-A and other members of the collectin family, the reader is referred to other recent reviews [4] [5] [6] .

SP-D is synthesized and secreted into the airspaces of the lung by the respiratory epithelium [1] . At the alveolar level, SP-D is constitutively synthesized and secreted by alveolar type II cells. More proximally in the lung, SP-D is secreted by a subset of bronchiolar epithelial cells, the non-ciliated Clara cells. Because SP-D is stored within the secretory granules of Clara cells [7, 8] , it seems likely that SP-D is subject to regulated secretion via granule exocy-tosis at this level of the respiratory tract. In some species, SP-D is also synthesized by epithelial cells and/or submucosal glands associated with the bronchi and trachea [9] . Although many alveolar macrophages show strong cytoplasmic and/or membrane labeling with antibody against SP-D, they do not contain detectable SP-D message.

The lung seems to be the major site of SP-D production. However, there is increasing evidence for extrapulmonary sites of expression as assessed with monoclonal or affinity-purified antibodies, reverse-transcriptase-mediated PCR (RT-PCR), and/or hybridization assays of tissues from humans and other large mammals [10 • ,11-14] (summarized in Table 1 ). It is difficult to entirely exclude crossreactions or amplification of related sequences; however, localization to many of these sites in human tissues was confirmed by using monoclonal antibodies in combination with RT-PCR with sequencing of the amplified products [10 • ]. Non-pulmonary expression seems to be largely restricted to cells lining epithelial surfaces or ducts and certain glandular epithelial cells that are in direct or indirect continuity with the environment. Notable exceptions to this generalization might include heart, brain, pancreatic islets, and testicular Leydig cells. SP-D has also been identified in amnionic epithelial cells by immunohistochemistry [15] ; however, it is unclear whether this is synthesized locally or derived from the lung by way of the amniotic fluid. Interestingly, in many of these sites SP-D microscopically co-localizes with gp-340, an SP-D binding protein and putative SP-D receptor [10 • ].

Sites of extrapulmonary expression have also been described in small mammals. In the rat, SP-D message was identified in RNA extracted from skin and blood vessel [16] , and both protein and message were identified in gastric mucosa [17] and mesentery [13] . Using RT-PCR, SP-D message has also been identified in mouse stomach, heart, and kidney [14] .

SP-D (43 kDa, reduced) consists of at least four discrete structural domains: a short, N-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide or coiled-coil neck domain, and a C-terminal, Ctype lectin carbohydrate recognition domain (CRD). Each molecule consists of trimeric subunits (3 × 43 kDa), which associate at their N-termini (Fig. 1) . Although most preparations of SP-D contain a predominance of dodecamers (that is, four trimeric subunits), the proportions of various oligomers vary between species. For example, rat lavage and recombinant rat SP-D are almost exclusively assembled as dodecamers (four trimers), whereas recombinant human SP-D is secreted as trimers, dodecamers and higher-order multimers [18] . SP-D isolated from the lavage of some patients with alveolar proteinosis consists predominantly of higher-order multimers, which can contain up to 32 (or more) trimeric subunits (Fig. 1 ).

Recent crystallographic and mutagenesis studies suggest that the structural determinants of saccharide binding are similar to those originally described for mannose-binding lectin [19,20,21 • ,22 • ]. At least two bound calcium ions and two intrachain disulfide crosslinks stabilize the required tertiary structure, and Glu321 and Asn323 within the CRD participate in glucose/mannose type recognition. Interactions with at least one glycolipid ligand, phosphatidylinositol (PI), require the participation of the C-terminal end of the protein [23, 24] .

A trimeric cluster of CRDs is necessary for high-affinity binding to carbohydrate ligands [21 • ,25]. The crystal structure of human SP-D suggests that the spatial distribution of CRDs within a trimeric subunit permits simultaneous and cooperative interactions with two or three glycoconjugates displayed on the surface of a particulate ligand [21 • ]. Furthermore, solid-phase binding studies have shown that monomeric CRDs have an approximately 10-fold lower binding affinity for multivalent ligands than trimeric CRDs.

Crystallographic studies of human SP-D further suggest that the spatial organization of CRDs within a trimer is stabilized by interactions of the C-terminal sequence with the trimeric neck domain [21 • ,26]. Interestingly, the three CRDs show a deviation from threefold asymmetry, suggesting some flexibility of the CRDs in relation to the neck. Thus, the dependence of the binding of PI on the C-terminal sequence could reflect conformational effects, rather than the direct participation of this sequence in ligand interactions.

The collagen domain length of SP-D is highly conserved and lacks interruptions in the repeating Gly-X-Y sequence (in which X and Y are different amino acids). As for other collagenous proteins, this domain is enriched in imino acids and contains hydroxyproline. Unlike SP-A, SP-D also contains hydroxylysine. Although the collagen domain of rat, human, bovine, and mouse SP-D lacks cysteine residues, cDNA sequencing has identified a codon for cysteine within the collagen domain of pig SP-D [27 • ]; this suggests the possibility of alternative patterns of chain association and oligomeric assembly for pig SP-D.

The first translated exon of SP-D contains a highly conserved and unusually hydrophilic Gly-X-Y sequence that shows little homology with the remainder of the collagen sequence. The functional significance of this region is unknown. However, it has been suggested that this region contributes to oligomer assembly or mediates interactions with cellular receptors.

The collagen domain determines the maximal spatial separation of trimeric, C-terminal lectin domains within SP-D molecules, but might also contribute to normal oligomeric assembly and secretion. For example, deletion of the entire collagen domain of rat SP-D results in the secretion of trimers rather than dodecamers [28] . In addition, 2,2-dipyridyl, an inhibitor of prolyl hydroxylation that interferes with the formation of a stable collagen helix, causes the intracellular accumulation of 43 kDa monomers and dimers [29] . In any case, the complete conservation of the number of Gly-X-Y triplets suggests that the spatial separation of trimeric CRDs is critical for normal SP-D function.

The N-terminal peptide of the mature protein contains two conserved cysteine residues at positions 15 and 20. These residues participate in interchain disulfide crosslinks that stabilize the trimer, as well as the N-terminal association of four or more trimeric subunits. Stable oligomerization of trimeric subunits permits cooperative or bridging interactions between spatially separated binding sites on the same surface or on different particles.

The process of forming interchain disulfide bonds is complex, and appropriate crosslinking of the N-terminal domains might be rate limiting for secretion [30] . Subcellular fractionation studies suggest that interchain bonds form initially between the three chains of a trimeric subunit. Subsequent rearrangements within the rough endoplasmic reticulum might allow the covalent crosslinking of a single chain from one subunit and two crosslinked chains of another, with the associated elimination of free thiol groups. Mutant proteins that contain unpaired N-terminal cysteine residues are not secreted. However, it is unclear whether this results from abnormalities in disulfide bonding itself, or the failure to stabilize the required N-terminal conformation.

The collagen domain contains hydroxylysyl-derived glycosides and a single N-linked oligosaccharide. In most species (human, rat, mouse, and cow) the site of N-linked glycosylation is located near the N-terminal end of the collagenous domain. Recently, it was shown that pig SP-D has an additional potential site of N-linked glycosylation within the CRD [27 • ]. Although rat and human lung lavage SP-D seem to be sialylated, as suggested by charge heterogeneity and cleavage with highly purified neuraminidase, preparations of human amniotic fluid and bovine lavage SP-D recovered from amniotic fluid showed predominantly complex type biantennary structures and no sialic acid [31] .

A variant form of SP-D (50 kDa) has been identified in lavage from a subset of human lavage samples; this protein shows O-linked glycosylation of threonyl residues within the N-terminal peptide domain [32 • ]. At present, the functional significance of these sugars is not known. The presence of O-linked glycosylation within the N-terminal domain might be predicted to interfere with normal dodecamer assembly. In this regard, the O-glycosylated 50 kDa form of human SP-D is recovered as trimeric subunits or smaller species.

As for many glycoproteins, the functional role of the attached carbohydrate is unknown. Mutational analysis has shown that the N-linked sugar on rat SP-D is not required for secretion, for dodecamer formation, or for interactions with a variety of microorganisms [29,33].

Consistent with its designation as a 'mannose-type' C-type lectin, SP-D preferentially binds to simple and complex saccharides containing mannose, glucose, or inositol [34, 35] . SP-D also interacts with specific constituents of pulmonary surfactant including PI [36-38] and glucosylceramide [39] . Binding to glucosylceramide involves interactions of the carbohydrate-binding sequences of the CRD with the glucosyl moiety. However, the interaction of SP-D with PI involves interactions with the lipid, as well as CRD-dependent interactions with the inositol moiety [24, 40] .

Microorganisms are surfaced with a diverse and complex array of polysaccharides and glycoconjugates, and most classes of microorganism contain one or more sugars recognized by SP-D. However, the outcome of this interaction depends on the specific organism and can be modified by the conditions of microbial growth. The potential consequences of this interaction include the following: varying degrees of lectin-dependent aggregation (namely, microbial agglutination), enhanced binding of microorganisms or microbial aggregates to their 'receptors' on host cells, phagocyte activation, and opsonic enhancement of phagocytosis and killing, potentially involving one or more cellular receptors for SP-D. Binding to organisms in suspension is often -but not always -accompanied by some degree of aggregation.

SP-D binds to purified lipopolysaccharide (LPS) isolated from a variety of Gram-negative organisms [35, 41] . In addition, LPS is the major cell wall component that is labeled on lectin blotting of outer membranes isolated from Escherichia coli [41] . The latter interactions involve the recognition of the core oligosaccharide domain, which contains glucose and heptose [41] . SP-D interacts preferentially with purified LPS molecules characterized by short or absent O-antigens and preferentially agglutinates bacterial strains expressing a predominance of rough (O-antigen-deficient) LPS [41, 44] .

Although the core oligosaccharide domain of LPS constitutes the major ligand for SP-D on at least some Gram-negative bacteria, the mechanism of interaction with this group of microorganisms is probably heterogeneous. SP-D binds to some smooth, unencapsulated strains of Gram-negative bacteria by immunofluorescence. The mechanism is uncertain; the quantity or quality of binding differs from that observed for rough strains and does not necessarily result in agglutination. LPS molecules on the surfaces of bacteria show heterogeneity in the extent of maturation, so it is possible that this interaction is mediated by a subpopulation of LPS with deficient O-antigens and that the density of binding sites is too low for high-affinity binding.

The recognition of the surface glycoconjugates on Gramnegative bacteria by SP-D depends not only on the expression of lectin-specific residues by a given strain or species, but also on the accessibility of these residues [1, 45] . For example, SP-D binds inefficiently to the core region of LPS of encapsulated Klebsiella, but efficiently agglutinates the corresponding unencapsulated phase variants. Interactions of SP-D with the core oligosaccharides of Gram-negative organisms are also influenced by the number of repeating saccharide units associated with the terminal O-antigen of the LPS [41,44].

Other potential ligands include the O-antigen domain of LPS, certain capsular polysaccharides, and membraneassociated glycoproteins. In this regard, SP-D can bind to di-mannose containing O-antigens expressed by a subset of Klebsiella serotypes (I Ofek, H Sahly and EC Crouch, unpublished data). Although other C-type lectins, specifically SP-A and the mannose receptor, can interact with specific capsular polysaccharides [46], a specific interaction of SP-D with capsular glycoconjugates or exopolysaccharides has not been described.

The mechanism of interaction with Gram-positive organisms has not been elucidated. Lipoteichoic acids, which are the major glycolipids associated with the Gram-positive cell wall, do not detectably compete with LPS for binding to SP-D (I Ofek, A Mesika, M Kalina, Y Keisari, D Chang, D McGregor and EC Crouch, manuscript submitted). In preliminary studies we observed that binding was competed only partly with maltose and/or EDTA, raising the possibility that binding might be more complex than for some Gram-negative organisms. . However, similar effects were observed when the neutrophils were preincubated with SP-D, and there was only a slight enhancement of uptake when bacteria were incubated with human SP-D and washed before their addition to neutrophils. Notably, the extent of binding and internalization was dependent on the extent of multimerization, with human SP-D multimers demonstrating the highest potency. Differences in cell type, the extent of SP-D multimerization, or differences in size or organization of bacterial aggregates could account for some of the apparent inconsistencies.

Although LPS mediates the binding of SP-D to at least some Gram-negative bacteria, SP-D can also bind to spe- In the latter study the authors suggested that fungal aggregation inhibits phagocytosis. Interestingly, SP-D binding directly inhibited fungal growth and decreased the outgrowth of pseudohyphae, the invasive form of the fungus, in the absence of phagocytic cells [57] . It is possible that these effects are also secondary to agglutination, possibly as result of nutrient deprivation.

Purified rat and human SP-D inhibit the infectivity and hemagglutination activity of influenza

SP-D can interact with host cells, both directly and indirectly. As indicated above, SP-D can enhance the phagocytosis and killing of certain microorganisms and enhance the oxidant response to microbial binding. However, at present there is only one study that suggests that the enhancement of phagocytosis by SP-D might involve the participation of an opsonic receptor. Furthermore, the enhanced uptake of IAV seems to be mediated by viral aggregation, with enhanced interactions of the virus with its natural receptors on the host cell.

In any case, SP-D can interact directly with host cells, and in some cases can influence their behavior. SP-D is chemotactic and haptotactic for neutrophils and certain mononuclear phagocytes [59 • ,67-69] and can elicit directional actin polymerization in alveolar macrophages [69] . In this regard, SP-D is considerably more potent than SP-A. Early studies with natural proteins isolated from silicotic animals reported directed effects on the oxidant metabolism of isolated alveolar macrophages [70] . However, such effects can probably be attributed to endotoxin contamination and/or aggregation. Purified dodecamers do not significantly increase the production of nitric oxide [71] or of proinflammatory cytokines such as tumor necrosis factor-α (Y Kesari, H Wang, A Mesika, E Crouch and I Ofek, unpublished data). Interestingly, purified SP-D has been reported to increase the production of several metalloproteinases in the absence of a significant effect on proinflammatory cytokine production [72] .

Despite the ability of SP-D to modulate a variety of cellular functions, little is currently known about potential cellular receptors for this protein. compartments [73] , but it is unclear whether the uptake is receptor dependent and whether SP-D is being internalized in association with specific ligands.

There are at least two classes of binding to host cells: CRD-dependent and CRD-independent. Some studies have demonstrated CRD-dependent binding to phagocytes that can be inhibited with EDTA or competing saccharides, both in vitro and in vivo.

As indicated above, the ability of SP-D to elicit the chemotaxis of neutrophilic and monocytic cells depends on the lectin activity of SP-D [68] . In addition, Kuan and coworkers reported that extracting formaldehyde-fixed alveolar macrophages with detergents largely eliminates the binding of purified SP-D, suggesting a membrane-associated ligand or glycolipid receptor [73] . Dong and Wright have extended these findings and suggest that PI can contribute to SP-D binding by alveolar macrophages [74] .

It is of interest that SP-D can bind to recombinant sCD14 through interactions with N-linked oligosaccharides [51 • ].

Given that the membrane-associated form of CD14 is widely expressed on host cells, it is possible that CD14 can serve as a binding site on macrophages and other cell types.

The phagocytic uptake of certain bacteria by neutrophils is also inhibited by calcium chelation or competing sugars [42]; however, this could result from the inhibition of microbial agglutination rather than lectin-dependent interactions with the phagocyte. Wang et al suggested that SP-D can bind to lymphocytic cells by a lectin-dependent mechanism [75 •• ] . In this regard, it is interesting to note that glucosylceramide, a ligand for SP-D in vitro, is one of the most abundant neutral glycolipids expressed by lymphoid cells.

Reid and co-workers were the first to present evidence for lectin-independent binding [76] . These and other studies suggested that binding does not involve known C1q or collectin receptors. The only putative receptor protein, gp-340, is a widely expressed member of the scavenger receptor superfamily [77,78 • ]. It binds to the CRD of SP-D in a calcium-dependent manner that does not require the lectin activity of SP-D. Although the protein has been immunolocalized to alveolar macrophage membranes and distributes together with SP-D in many different human tissues [10 • ,77], it has not yet been shown to mediate the binding of SP-D to these cells or to participate in signal transduction events. The cDNAs isolated from lung have not shown a membrane-spanning region [77] , and the protein is abundant as a soluble component in BAL. Given that gp-340 is a highly multimerized protein that contains numerous potential ligand binding domains (Fig. 1b) , it is possible that the protein cooperates with SP-D in the neu-tralization or clearance of certain ligands rather than specifically mediating the interactions of SP-D with host cells.

Wright and co-workers have demonstrated the binding of SP-D to isolated type II pneumocytes. The mechanism seems distinct from the binding to macrophages [79 • ]. The binding was dependent on concentration, time, and temperature and required calcium; it was not sensitive to protease treatment or to PI-phospholipase C. Although the internalized SP-D was degraded or recycled to lamellar bodies, SP-D binding did not alter the uptake of surfactant lipids.

SP-D has demonstrated comparatively few direct effects on the metabolism of host cells, at least in situations where self-aggregation and endotoxin contamination have been excluded. One possible explanation is that modulation of cellular function requires the prior interaction of SP-D with a ligand. This would have numerous potential physiological advantages, because the presence of 'active' protein might be restricted to sites of microbial or antigenic deposition. The binding of complex, multivalent, particulate antigens to two or more CRDs could markedly alter the conformation of SP-D molecules, with respect to the spatial orientation of the arms in relation to the N-terminal crosslinking domain and/or with respect to the spatial orientation of the CRDs within a given trimeric subunit. Thus, the 'charging' of SP-D with a particulate ligand could lead to local or distant conformational changes that expose 'cryptic' binding sites for cellular receptors.

There is some preliminary evidence consistent with the notion that the interaction of SP-D with a ligand alters its capacity to activate host cells. Table 3 and discussed below.

SP-D can be isolated in different multimeric forms from proteinosis lavage [32 • ] and are produced by Chinese hamster ovary K1 cells transfected with human SP-D cDNA [18] . As described previously, the effects of SP-D on the neutrophil response to influenza virus are highly dependent on the ability of SP-D to agglutinate the viral particles, and the agglutination activity is directly correlated with the extent of multimerization. Trimers can bind to the virus but have little capacity to modulate neutrophil interactions. By contrast, highly multimerized proteins show greater activity than dodecamers [81] .

Given these observations, factors that favor enhanced oligomerization or lead to the accumulation of trimeric subunits promote might influence SP-D function. For example, the liberation of active trimers by a hypothetical microbial protease could lead to the accumulation of molecules that might inhibit the aggregation-dependent activities of SP-D.

In contrast, recombinant trimeric CRDs can stimulate chemotaxis [67] and decrease viral infectivity [65 • ]. Although higher-order oligomers of SP-D can self-aggregate and precipitate in the presence of calcium in vitro, the functional consequences are not known.

The lectin activity of SP-A is decreased after the nitric oxide-dependent nitration of tyrosine residues [82] , and nitration decreases the ability of SP-A to enhance the adherence of Pneumocystis to alveolar macrophages [83] . However, similar findings have not yet been reported for SP-D. Conditions of mildly acidic pH, as might be found in endocytic compartments, are predicted to disrupt the lectin-dependent activities of SP-D [34]. Proteolytic degradation remains an important possibility. However, SP-D is highly resistant to degradation by a wide variety of neutral proteases in vitro, and degradation products have not yet been shown to accumulate under pathological conditions in vivo.

Glucose concentrations at levels encountered in diabetes can interfere with SP-D's ability to interact with specific strains of IAV or other microorganisms in vitro [84 • ]. Many microorganisms release cell wall polysaccharides or glycoconjugates, which might interfere with the binding of collectins to the same or other organisms. In this regard, SP-D recovered from rats after the instillation of LPS into the airway shows decreased lectin activity, which is attributed to occupancy of the CRD with LPS [49 • ]. It seems reasonable to speculate that some organisms might compete with other organisms for binding to SP-D. Such a situation could conceivably predispose to secondary infections. Lastly, the potential inhibitory effects of competing saccharide ligands presents important methodological considerations for experiments using carbohydrate-containing cell culture medium or buffers. Non C-type lectins (such as ficolins)

It is difficult to predict the functions of SP-D within the airspace. Other lectins with overlapping specificity are also present. Although the levels of mannose-binding lectin are probably low in the absence of increased vascular permeability, SP-A and the macrophage mannose receptor could conceivably interact with the same ligands in the distal airways and alveoli. Such interactions could lead to antagonistic or cooperative effects. Furthermore, we have little knowledge regarding the microanatomic distribution of these molecules in specific circumstances in vivo.

Although most SP-A is probably associated with the insoluble phase of the alveolar lining material, and the macrophage mannose receptor is membrane-associated, the distribution might be altered in the setting of lung injury.

Models of SP-D deficiency show no detectable anatomical or physiological abnormalities at birth. However, the animals gradually develop a patchy, subpleural alveolar lipidosis with associated type II cell hypertrophy, the accumulation of enlarged and foamy macrophages, and an apparent expansion of peribronchial lymphoid tissue [85 • ,86 • ]. Interestingly, the mice eventually develop distal-acinar emphysema and areas of subpleural fibrosis, which could reflect a continuing inflammatory reaction associated with abnormal oxidant metabolism and metalloproteinase activity [87 • ]. By contrast, SP-A-deficient mice (-/-) show essentially normal respiratory function and surfactant lipid metabolism [88, 89] but numerous apparent host defense abnormalities [90] .

The capacity of SP-D to bind to specific strains of influenza A in vitro is highly correlated with the capacity of the virus to proliferate in mice in vivo [62] . Specifically, strains with more oligosaccharide attachments on the HA are preferentially neutralized by SP-D in vitro and show decreased proliferation in mice. Because the administration of mannan together with the virus increased the replication of IAV in the lung, the involvement of a mannose-type, C-type lectin was implicated.

SP-D-sensitive IAV strains also replicate to higher titers in the lungs of diabetic mice than in nondiabetic controls [84 • ]. Replication of the virus is positively correlated with blood glucose level, and decreases in response to insulin treatment. Significantly, blood glucose levels comparable to those measured in the diabetic mice were sufficient to inhibit the interaction of SP-D with these viral strains in vitro. PR-8, a strain that does not interact with SP-D but does interact with SP-A, replicated to the same extent in diabetic and control mice.

SP-D levels increase in association with certain infections. For example, SP-D levels, but not the levels of serum mannose-binding lectin, increase markedly after IAV infection [62] . Impressive increases in SP-D have also been observed in murine models of Pneumocystis carinii [91] and P. aeruginosa infection [92] .

SP-D-deficient mice have not yet been extensively characterized with respect to host defense function. However, they show decreased viral clearance and enhanced inflammation after challenge with respiratory syncytial virus [93] and IAV (AM Levine, personal communication). In addition, they show increased inflammation, increased oxidant production, and decreased macrophage phagocytosis in response to intratracheally instilled group B streptococcus and Haemophilus influenzae (AM Levine, personal communication). Although the overexpression of wild-type SP-D in type II pneumocytes with the SP-D-deficient mice can prevent the lipidosis and inflammatory changes [94] , the ability of overexpressed wild-type SP-D or exogenous SP-D to ameliorate these abnormalities has not yet been described. The coexisting pulmonary abnormalities also complicate the interpretation of challenge models. For example, macrophage activation might enhance killing and offset any decrease that results more directly from SP-D deficiency. SP-D deficiency modifies the host response to instilled LPS with decreased lung injury and inflammatory cell recruitment [50].

Molecules that can bind to potential antigens and deliver them to macrophages and other antigen-presenting cells might contribute to the development of acquired immunity. In this regard, a few published observations suggest possible roles in the development of humoral and/or cellular immunity in response to microorganisms or complex organic antigens. For example, SP-D can decrease interleukin-2dependent T-lymphocyte proliferation [95 • ]. Interestingly, single-arm mutants were at least as potent as intact dodecamers in mediating this effect. SP-D also binds to oligosaccharides associated with dust mite allergen [96 • ], and can inhibit the binding of specific IgE to these allergens, possibly through direct, CRD-dependent binding to lymphocytes [96 • ]. Thus, alterations in the level of SP-D (or the state of oligomerization) might influence the development of immunological responses and contribute to the pathogenesis of asthma and other hypersensitivity disorders.

There are other potential interplays between humoral immunity and collectins with regard to antimicrobial host defense. For example, increased glycosylation of IAV coat proteins, an adaptation that is believed to help the virus to evade antibody-mediated neutralization, is associated with increased reactivity with SP-D and other collectins [62]. Thus, the relative potential importance of antibody and collectin-mediated host defenses might be influenced by subtle variations in the structure of the microbial surface.

There is little recent information on the developmental regulation of SP-D expression. In general, SP-D increases rapidly late in gestation [97] [98] [99] [100] . The production of SP-D increases during the culture of fetal lung explants, and expression can be increased with glucocorticoids [98, 100, 101] . The exposure of fetal rats to glucocorticoids in vivo leads to precocious expression with increased numbers of SP-D-expressing cells and increased cellular levels of SP-D message [98, 101, 102] .

Although SP-D is produced constitutively within the lung, protein accumulation and gene expression are inducible and increases in SP-D expression have been observed in a number of disease states or models (Tables 4 and 5 ). In general, the synthesis and secretion of SP-D increase in association with lung injury and activation of the respiratory epithelium [1] . For example, levels of SP-D mRNA and SP-D accumulation are increased within 24-72 h after intratracheal instillation of LPS [103 • ], and SP-D expression by alveolar and bronchiolar epithelial cells increases after exposure of rats to 95% O 2 for 12 h [104] . Keratinocyte growth factor (KGF) increases SP-D expression and protein production in association with pneumocyte hyperplasia and after injury caused by bleomycin [105] . In addition, the levels of SP-D can increase markedly in response to the overexpression of certain cytokines, such as interleukin-4, or in response to microbial challenge [91, 92] .

Studies of the upstream regulatory region of the SP-D gene have demonstrated increased promoter activity in the presence of glucocorticoids, which is consistent with the findings in vivo and in lung organ culture [106] . However, no functional glucocorticoid response elements have been identified, and the effects of dexamethasone seem to be secondary and involve the effects of other transregulatory molecules.

The activity of the human SP-D promoter is dependent on a conserved activator protein-1 (AP-1) element (-109) that binds to members of the fos and jun families of transcriptional factors [107] . In addition, the promoter contains multiple functional binding sites for CCAAT-enhancer-binding protein (C/EBP) transcription factors. Mutagenesis experiments suggest that these are required for basal and stimulated promoter activity, and promoter activity is markedly increased in H441 cells after co-transfection with C/EBPβ cDNA (YC He and E crouch, unpublished data). The importance of the conserved AP-1 element and the presence of multiple binding sites for C/EBP transcription factors is consistent with the observed modulation of SP-D expression in the setting of tissue injury.

SP-D promoter activity is not dependent on the binding of thyroid transcription factor 1 (TTF-1) [107] . However, promoter activity is dependent on two interacting forkhead binding sites, upstream and downstream of the AP-1 element; these sites bind to hepatic nuclear factor-3α and apparently other forkhead box proteins in H441 lung adenocarcinoma nuclear extracts [107] .

Initial comparison of genomic and cDNA sequence suggested the existence of genetic polymorphisms in the SP-D coding sequence, including one in the N-terminal propeptide domain (Thr11 compared with Met11 in the mature protein) and three additional differences within the collagen domain at positions 102, 160, and 186 [108] . The latter substitutions are conservative to the extent that they are not expected to disrupt the collagen helix. Floros Table 5 Increased SP-D accumulation or expression in animal models

Silicosis Rat [118] Hyperoxia Rat [104] Endotoxin (LPS) Rat [103] Challenge with P. aeruginosa Mouse [92] Challenge with IAV Mouse

[62]

Challenge with Pneumocystis carinii SCID mouse [91] Rat [119] Overexpression of interleukin-4 Mouse [120] SCID, severe combined immunodeficiency. and co-workers have recently confirmed the existence of polymorphisms at positions 11 and 160 of the mature protein [109] . The potential biological significance, if any, is not known. Interestingly, the 50 kDa variant of SP-D showed O-linked glycosylation of Thr11 [32 • ], suggesting that this polymorphism might be associated with altered glycosylation. Interestingly, the 50 kDa variant was recovered as trimeric subunits, raising the possibility that differences in the glycosylation of residue 11, which is immediately N-terminal to Cys15, could influence multimerization and the capacity of SP-D to participate in bridging interactions.

There is increasing evidence that SP-D interacts specifically with a wide variety of respiratory pathogens, modulates the leukocyte response to these organisms, and participates in aspects of pulmonary immune and inflammatory regulation (Table 6) . SP-D can influence the activity of phagocytes through CRD-dependent and CRD-independent interactions. At least some of the effects of SP-D result from aggregation with enhanced binding of the agglutinated ligand to their natural 'receptors'. Although the lung is the major site of SP-D expression, it is likely that the protein has more generalized roles in host defense and the acute response to infection and tissue injury. 16 Role of endothelin-1 in lung disease Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease. from Xenopus laevis [16] . ETA receptors in normal lung are found in greatest abundance on vascular and airway smooth muscle, whereas ETB receptors are most often found on the endothelium. Clearance of ET-1 from the circulation is mediated by the ETB receptor primarily in the lung, but also in the kidney and liver [17] .

Activation of both ETA and ETB receptors on smooth muscle cells leads to vasoconstriction whereas ETB receptor activation leads to bronchoconstriction. Activation of ETB receptors located on endothelial cells leads to vasodilation by increasing nitric oxide (NO) production. The mitogenic and inflammatory modulator functions of ET-1 are primarily mediated by ETA receptor activity. Binding of the ligand to its receptor results in coupling of cell-specific G proteins that activate or inhibit adenylate cyclase, stimulate phosphatidyl-inositol-specific phosholipase, open voltage gated calcium and potassium channels, and so on. The varied effects of ET-1 receptor activation thus depend on the G protein and signal transduction pathways active in the cell of interest [18] . A growing number of receptor antagonists exist with variable selectivity for one or both receptor subtypes.

Regulation of ET-1 is at the level of transcription, with stimuli including shear stress, hypoxia, cytokines (IL-2, IL-1β, tumor necrosis factor α, IFN-β, etc), lipopolysaccharides, and many growth factors (transforming growth factor-β, platelet-derived growth factor, epidermal growth factor, etc) inducing transcription of ET-1 mRNA and secretion of protein [18] . ET-1 acting in an autocrine fashion may also increase ET-1 expression [19] . ET-1 expression is decreased by NO [20] . Some stimuli may additionally enhance preproET-1 mRNA stability, leading to increased and sustained ET-1 expression. The number of ETA and ETB receptors is also cell specific and regulated by a variety of growth factors [18] . Because ET-1 and receptor expression is influenced by many diverse physical and biochemical mechanisms, the role of ET-1 in pathologic states has been difficult to define, and these are addressed in subsequent parts of this article.

In the airway, ET-1 is localized primarily to the bronchial smooth muscle with low expression in the epithelium. Cellular subsets of the epithelium that secrete ET-1 include mucous cells, serous cells, and Clara cells [21] . ET binding sites are found on bronchial smooth muscle, alveolar septae, endothelial cells, and parasympathetic ganglia [22, 23] . ET-1 expression in the airways, as previously noted, is regulated by inflammatory mediators. Eosinophilic airway inflammation, as may be seen in severe asthma, is associated with increased ET-1 levels in the lung [24] . ET-1 secretion may also act in an autocrine or paracrine fashion, via the ETA receptor, leading to increased transepithelial potential difference and ciliary beat frequency, and to exerting mitogenic effects on airway epithelium and smooth muscle cells [25] [26] [27] [28] .

All three endothelins cause bronchoconstriction in intact airways, with ET-1 being the most potent. Denuded bronchi constrict equally to all three endothelins, suggesting considerable modulation of ET-1 effects by the epithelium [29] . The vast majority of ET-1 binding sites on bronchial smooth muscle are ETB receptors, and bronchoconstriction in human bronchi is not inhibited by ETA antagonists but augmented by ETB receptor agonists [30] [31] [32] . Since cultured airway epithelium secretes equal amounts of ET-1 and ET-3, which have equivalent affinity for the ETB receptor, bronchoconstriction could be mediated by both endothelins [33] .

While ET-1 stimulates release of multiple cytokines important in airway inflammation, it does not enhance secretion of histamine or leukotrienes. ET-1 does increase prostaglandin release [32] . Inhibition of cyclo-oxygenase, however, has no effect on bronchoconstriction suggesting that, despite the release of multiple mediators, ET-1 mediated bronchoconstriction is a direct effect of activation of the ETB receptor [32] . ETA mediated bronchoconstriction may also be important following ETB receptor desensitization or denudation of the airway epithelium, as may occur during airway inflammation and during the late, sustained airway response to inhaled antigens [31, 34, 35] . Interestingly, heterozygous ET-1 knockout mice, with a 50% reduction in ET-1 peptide, have airway hyperresponsiveness but not remodeling, suggesting the decrease in ET-1 modulates bronchoconstriction activity by a functional mechanism, possibly by decreasing basal NO production [36, 37] .

Asthma is also an inflammatory airway disease characterized by bronchoconstriction and hyperreactivity with influx of inflammatory cells, mucus production, edema, and airway thickening. ET-1 may have important roles in each of these processes. While ET-1 causes immediate bronchoconstriction [38] , it also increases bronchial reactivity to inhaled antigens [35] as well as influx of inflammatory cells [39, 40] , increased cytokine production [40] , airway edema [41] , and airway remodeling [28, 42, 43] . Airway inflammation also leads to increased ET-1 synthesis, possibly perpetuating the inflammation and bronchoconstriction [44] . ET-1 release from cultured peripheral mononuclear and bronchial epithelial cells from asthmatics is also increased [45, 46] . Inhibition of ETA or combined ETA and ETB receptors additionally leads to decreased airway inflammation in antigen-challenged animals, suggesting that the proinflammatory effects of ET-1 in the airway are mediated by ETA receptors [39, 47] .

Children with asthma have increased circulating levels of ET-1 [48] . Adult asthmatics have normal levels between attacks but, during acute attacks, have elevated serum ET-1 levels that correlate inversely with airflow measurements and decrease with treatment [49] . Bronchoalveolar lavage (BAL) ET-1 in asthmatics is similarly increased to concentrations that cause bronchoconstriction and inversely correlates with forced expiratory volume in 1 s (FEV 1 ) [29, 50, 51] . As in cultured epithelial cells, ET-1 and ET-3 are found in equal amounts in BAL fluid from asthmatics [33, 52] . There is also a relative increase in ETB versus ETA receptor expression in asthmatic patients, which may contribute to increased bronchoconstriction [53] . Not all asthmatics, however, have increased ET-1 as patients with nocturnal asthma have decreased BAL ET-1 levels [54] . Treatment of acute asthma exacerbations with steroids, beta-adrenergic agonists or phosphodiesterase inhibitors resulted in decreased BAL ET-1 [52, 55] . Immunostaining and in situ hybridization for ET-1 in biopsy specimens from asthmatics have shown an increase in ET-1 in the bronchial epithelium that correlates with asthma symptoms [46, 56] .

Cigarette smoking leads to increased circulating ET-1 [57] but patients with chronic obstructive pulmonary disease, in the absence of pulmonary hypertension and hypoxemia, do not have increased plasma ET-1 [58] [59] [60] . Increases in urinary ET-1 instead correlate with decreases in oxygenation, possibly through hypoxic release of ET-1 from the kidney [61, 62] . Smokers also have impaired ET-1 mediated vasodilation that correlates with bronchial hyperresponsiveness and may contribute to pulmonary hypertension [63, 64] .

ET-1 has been implicated in the pathogenesis of bronchiectasis by its ability to promote neutrophil chemotaxis, adherence, and activation [65] [66] [67] [68] [69] . Sputum ET-1 levels are increased in patients with cystic fibrosis [59] , and sputum ET-1 correlated with Pseduomonas infection in noncystic fibrosis related bronchiectasis [70] .

ET-1 has also been implicated in the pathogenesis of bronchiolitis obliterans (BO), which is characterized by injury to small conducting airways resulting in formation of proliferative, collagen rich tissue obliterating airway architecture. BO is the leading cause of late mortality from lung transplantation, and ET-1 is increased in lung allografts [71] . The pro-inflammatory and mitogenic properties of ET-1 in the airways has led to speculation that ET-1 may be involved in formation of the lesion [28] . This is further supported by the increase in BAL ET-1 in lung allografts [72, 73] . The in vivo gene transfer of ET-1 to the airway epithelium using the hemagglutinating virus of Japan in rats recently resulted in pathologic changes in the distal airways identical to those seen in human BO specimens [74] . These changes were not due to nonspecific effects of the hemagglutinating virus of Japan itself, but could be attributed to the presence of the ET-1 gene, which was localized to the airway epithelium, hyperplastic lesions, and alveolar cells.

Pulmonary hypertension is a rare and progressive disease characterized by increases in normally low pulmonary vascular tone, pulmonary vascular remodeling, and progressive right heart failure. ET-1 has been implicated as a mediator in the changes seen in pulmonary hypertension. In the pulmonary vasculature, ET-1 is found primarily in endothelial cells and to a lesser extent in the vascular smooth muscle cells. The endothelium secretes ET-1 primarily to the basolateral surface of the cell. ET-1 secretion may be increased by a variety of stimuli including cytokines, catecholamines, and physical forces such as shear stress, and decreased by NO, prostaglandins, and oxidant stress [20, [75] [76] [77] [78] . Hypoxia has been reported to increase, have no effect, or decrease ET-1 release from endothelial cells [79] [80] [81] [82] [83] .

Activation of the receptors for ET-1 in the pulmonary vasculature leads to both vasodilation and vasoconstriction, and depends on both cell type and receptor. In the whole lung, ETA receptors are the most abundant and are localized to the medial layer of the arteries, decreasing in intensity in the peripheral circulation [84, 85] . ETB receptors are also found in the media of the pulmonary vessels, increasing in intensity in the distal circulation, while intimal ETB receptors are localized in the larger elastic arteries [85] . This distribution of receptors has important implications in understanding ET-1 regulation of vascular tone. Vascular ET-1 receptors may be increased by several factors including angiotensin and hypoxia [80, [85] [86] [87] .

ET-1 can act as both a vasodilator and vasoconstrictor in the pulmonary circulation. Generation of NO or opening of ATP-sensitive potassium channels leading to hyperpolarization results in vasodilation mediated by ETB receptors on pulmonary endothelium [88, 89] . In hypertensive, chronically hypoxic lungs with increased ETB receptor expression, augmented vasodilation is due to increased ETB mediated NO release that is inhibited by hypoxic ventilation, while inhibition of NO synthesis leads to increased ET-1 mediated vasoconstriction [85, [90] [91] [92] . Both ETA and ETB receptors, conversely, acting on vascular smooth muscle, mediate ET-1 induced vasoconstriction. In the normal lung, ET-1 causes vasoconstriction primarily by activation of the ETA receptors in the large, conducting vessels of the lung [93, 94] . In the smaller, resistance vessels of the lung, ETB receptors in the media predominate and are responsible for the ET-1 induced vasoconstriction [93] . Interestingly, preconstriction of the pulmonary circulation resulted in a shift from primarily ETA mediated to ETB mediated vasoconstriction [94] .

The overall effect of ET-1 on vascular tone depends on both the dose and on the pre-existing tone in the lung. ET-1 administration during acute hypoxic vasoconstriction will result in transient pulmonary vasodilation [89] . This effect is dose dependent, with lower doses leading to vasodilation while higher or repetitive doses cause vasoconstriction following an initial, brief vasodilation [89] . The role of ET-1 in the acute hypoxic vasoconstriction in the lung is not certain. ETA receptor antagonism attenuates hypoxic pulmonary vasoconstriction in several species [95] , and ET-1 may be implicated in the mechanism of acute hypoxic response by inhibition of K-ATP channels [96] .