il-8 also possesses a clear role in eosinophil chemotaxis in allergic respiratory diseases [ 2 , 3 ] . on the other hand , il-15 effect on human eosinophil is through inhibition of the spontaneous apoptosis via the autocrine production of gm - csf and thus may perpetuate allergic inflammation by prolonged eosinophil survival .
nonetheless , the role of il-15 in the pathophysiology of allergic airways diseases is still in its infancy and remains a controversial issue [ 59 ] .
nk cells activation plays a pivotal role in viruses and tumor lyses ; however , they also produce cytokines and thus are thought to play a proinflammatory role .
the secretagogue activity of nk cells includes ifn- , il-10 , tnf- , mip-1 , mip-1 , and gm - csf .
these cytokines are augmented by il-15 , a cytokine produced by activated monocytes / macrophages [ 12 , 13 ] .
some investigators demonstrated the ability of nk cells to differentiate in the presence of il-4 into nk cell subsets secreting distinct cytokines patterns similar to th2 profile such as il-5 and il-13 [ 14 , 15 ] .
a more recent study demonstrated the existence of type 2 cytokine - secreting nk cells in ar and showed increased number and enhanced cytotoxicity of nk cells .
nonetheless , whether nk cells are able to attract eosinophils through il-8 secretion is not known .
increasing evidence supports a novel immunoregulatory role for vitamin d in allergic diseases such as asthma and ar [ 17 , 18 ] .
the ability of vitamin d to reduce cytokines from inflammatory cells is also well acknowledged .
therefore , the therapeutic potential of vitamin d as an anti - inflammatory agent in allergic diseases remains a subject of interest .
herein , we show a novel crosstalk between human nk cell and eosinophil recruitment through il-15/il-8 axis .
results are discussed in relation to the possible novel therapeutic effect of vitamin d in downregulating this inflammatory event , and the possible role for nk cells in the pathophysiology of ar .
nk cells were isolated from buffy coats to obtain large amount of cells for culture purposes , and eosinophils were freshly isolated from the peripheral blood of sixteen ar patients sensitized to different aeroallergens that were confirmed by skin tests and/or radioallergosorbent test ( rast ) .
eosinophils and nk cells were further purified by negative selection immunomagnetic cell separation ( macs ; miltenyi biotec , bergisch gladbach , germany ) , using anti - cd16 magnetic beads for eosinophils purification , and a cocktail of biotin - conjugated antibodies against lineage - specific antigens and a cocktail of microbeads ( nk cell isolation kit - human , miltenyi biotec , bergisch gladbach , germany ) for nk ( cd56+ve cd3-ve ) cells purification .
nk cells ( 1  10 cells ) were incubated in the presence or absence of 1  ng / ml il-15 ( r&d systems ; minneapolis , mn ) , 10  m concentration of vitamin d3 ( cayman chemical ) , 10  m concentration of h-89 dihydrochloride ( vwr - calbiochem ) , a pka inhibitor , 10  m concentration of bisindolylmaleimide ( vwr - calbiochem ) , a pkc inhibitor , 10  m concentration of sb203580 ( vwr - calbiochem ) , a p38 map - kinase inhibitor , or 1  m concentration of genistein ( sigma ) , a tyrosine kinase inhibitor , at 37c-5% co2 with 1  ml rpmi-1640 supplemented with 100  u  penicillin / ml and 100  g streptomycin / ml ( lonza , verviers , belgium ) , 10% of inactivated fetal calf serum ( lonza ) in 24 wells plate ( bd biosciences ) . after 72  h incubation period ,
supernatants were carefully collected and analysed for their eosinophilotactic activity or il-8 content .   as for the measurement of il-8 content induced by peripheral blood nk cells from ar patients before and after nasal challenge , peripheral blood nk cells were purified from additional four ar patients to house dust mite ( hdm ) . after obtaining the patients consent , 30  mls of peripheral blood was obtained before and 6 hours after nasal challenge with hdm allergen .
purified nk cells at the concentration of 3  10  cells / ml were incubated in the culture medium for 72  hrs .
chemotaxis assays were performed in triplicate in a 48-well microchemotaxis boyden chambers incubated in 5% co2 at 37c for 90  min .
aliquots of 29  l of the nk supernatant were placed in the lower wells and 50  l of eosinophils suspension ( 10  cells / ml ) were placed in the upper wells .
the two chambers were separated by a 5.0  m pore polycarbonate membrane ( nuclepore , whatman , middlesex , uk )
. migrated cells adherent to the lower surface were counted in 5 selected high power fields / well under a light microscope ( 5  hpf ; x400 ) . as for blocking experiments , nk supernatants were preincubated for 1  h and during the chemotaxis assay , with different doses ( 1010  g / ml ) of the monoclonal anti - il-8ab that neutralizes the biological activity of il-8 ( r&d systems ; minneapolis .
the content of il-8 was measured in supernatants of nk cells using human il-8 cytoset kit ( invitrogen corporation , usa ) according to the manufacturers ' recommendations .
nk cells treatment with the optimal dose of 1  ng / ml il-15 during the culture period resulted in significant augmentation of nk cells supernatant - induced eosinophil chemotaxis from ar patients to almost 3-fold chemotaxis index .
these results indicate a novel autocrine activity for nk cells in recruiting eosinophils and highlight a novel role for il-15 in up - regulating this effect . 

figure 2 shows that eosinophil chemotaxis of ar patients against nk supernatant of cells treated by il-15 was significantly blocked by anti - il-8 ab .
the blocking activity was in a dose - dependent fashion , but was not completely blocked to the control level .
these results indicate that nk cells stimulated by il-15 secrete eosinophilotactic chemokines that include at least in part il-8 among others . to confirm this , we next measured by elisa the amount of il-8 spontaneously secreted by nk cells , and the modulatory effects of il-15 and vitamin d3 on il-8 secretion .  
we tested the amount of il-8 secreted by nk cells after 72  h culture period in the presence and absence of the optimal doses of il-15 or vitamin d3 .
as can be seen in table 1 , cultured nk cells for 72  h secreted il-8 .
the amount of recovered il-8 was increased from 88.64  21.5 to 178.9  23.6  pg / ml and was significantly reduced by vitamin d3 to 59.2  16.3  pg / ml .
these results indicate the ability of il-15 to upregulate the il-8 secretagogue activity by nk cells , and the ability of vitamin d3 to significantly inhibit the il-8 secretagogue activity of nk cells .
furthermore , it confirms the contribution of il-8 to the eosinophilotactic cytokines secreted by nk cells .   however , to gain insight into the possible signal pathway involved in nk cell induced - il-8 modulation by il-15 and vitamin d3 , we treated nk cells with different kinases inhibitors during the culture period .
dihydrochloride , bisindo - lylmaleimide , or genistein did not modulate the amount of il-8 secreted by nk cells ( data not shown ) .
however , as can be seen in table 1 , il-8 secretion by nk cells was sensitive to sb203580 and resulted in reduction of il-8 to 15  0.5  pg / ml .
similarly , the augmentation of il-8 secretion by il-15 was reduced by sb203580 to 54.75  5.7 .
these results may indicate the involvement of p38 map - kinase pathway in the signal transduction of il-8 secretion by nk cells . finally to further correlate our results to the pathophysiology of ar , we challenged ar patients to hdm with hdm allergen .
nk cells purification before and at 6  h postchallenge were cultured for 72  h. collected supernatants were checked by elisa for the amount of il-8 recovered . as demonstrated in figure 3 , after nasal challenge nk cells secreted a significantly higher amount of il-8 .
this result indicates the importance of nk cells as a source for il-8 in the pathophysiology of ar .
in the current communication , we demonstrated a novel crosstalk between nk cells and eosinophils via il-15/il-8 axis .
this may indicate a role for il-15 in the pathophysiology of allergic rhinitis late - phase reaction and the promotion of eosinophilic inflammation .   in ar disease ,
the mixed in vivo milieu of th2 cytokines especially induced after allergen exposure prime eosinophils .
herein , we show that nasal allergen challenge of ar patients also primes nk cells to secrete larger amounts of il-8 .
further , the ability of nk cells to secrete il-8 in their supernatants that contributed to the attraction of eosinophils from ar patients may provide a further explanation to eosinophil recruitment in ar .  
il-15 prolongs eosinophil survival and thus acts as a proeosinophilic inflammatory mediator . in the current study
, il-15 demonstrated a further novel indirect role in helping eosinophil recruitment , at least in part through secretion of il-8 from resting nk cells .  
vitamin d binds to its nuclear receptor , the vitamin d receptor ( vdr ) through which it signals in its target cells .
we demonstrated a significant inhibition of il-8 by nk cells in the presence of vitamin d3 .
this is consistent with an earlier study that demonstrated that vitamin d3 repressed il-8 promoter activity induced by tnf- in human melanoma cell line by 50% compared to 30% inhibition by dexamethasone , as well as tnf--induced il-8 release and il-8 mrna level .
the fact that il-8 secretion by nk cells was sensitive to p38 map - kinase inhibition may indicate that il-15 and vitamin d3 modulated il-8 autocrine activity of nk cells through the same pathway .
it is then reasonable to assume that il-15 may induce il-8 secretion by nk cells through stimulation of p38 map - kinase activity and that vitamin d3 reduces il-8 secretion from nk cells through blocking effect on the p38 map - kinase pathway .
, our results may indicate the following : a novel role for nk cells in the pathophysiology of ar through recruiting eosinophils ,   a novel modulatory role for il-15 in inducing eosinophils chemotaxis from ar patients through the nk cells secretagogue activity ,   identifying il-8 as an important cytokine that contributes to the eosinophilotactic agents secreted by nk cells ,   indicating that vitamin d3 may be an effective therapeutic modality .  
				
our results open channels for other researchers to further elaborate on the exact mechanism(s ) involved in nk - induced il-8 modulation by il-15 and vitamin d3 and p38 map - kinase inhibition .
 natural killer cells ( nk ) secrete eosinophilotactic cytokines , however , whether they contribute to eosinophil chemotaxis by secreting il-8 is not known . 
 we investigated the ability of cd56+cd3-ve ( nk cells ) to induce chemotaxis of peripheral blood eosinophils from allergic rhinitis ( ar ) patients , through il-8 secretion , and the effects of il-15 , the nk cell proactivating cytokine , and calcitriol : 1 , 25-dihydroxy vitamin d3 ( vitamin d3 ) , the immunomodulator agent , in this scenario . 
 herein , it is shown that supernatants from unstimulated nk cells exhibited chemotactic activity against eosinophil . 
 this effect was significantly augmented by il-15 ( 1  ng / ml ) treatment , resulting in an increase in the chemotactic index of approximately 3 folds and was abrogated by neutralizing antibody ( ab ) to il-8 in a dose - dependent fashion . the amount of il-8 secreted by nk cells was increased by il-15 treatment from levels of 88.64  21.5 to 178.9  23.6  pg / ml and was significantly reduced by 106  m vitamin d3 to levels of 59.2  16.3  pg / ml . our results indicate a novel inflammatory crosstalk between nk cells and eosinophils via il-15/il-8 axis that can be modulated by vitamin d3 . 