extfieldsofstudy: Biology Medicine

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Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation.

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.

The signaling events that mediate either proliferation or differentiation in cells of the macrophage lineage are poorly understood. Previously, the function of the CSF-1R in growth and differentiation has been examined by transfecting the CSF-1R into the murine hemopoietic FDC-P1 cells (FD) (16). These cells became responsive to CSF-1 and underwent changes in morphology suggestive of macrophage differentiation (17). Single and multiple substitutions of tyrosines for phenylalanines in the CSF-1R at positions 697 (FD/697), 706 (FD/706), and 721 (FD/ 721) were not observed to affect proliferation or differentiation (16). This lack of an effect on proliferation or differentiation occurred despite the inhibitory effects of the Y697F mutation on Grb-2 binding to the CSF-1R (18), the Y721F mutation on PI 3-kinase activation (9,19), and the Y706F mutation on Stat-1 activation (14). The Y807F substitution was observed to impair phospholipase C␥-2 activation, which appears to act with PI 3-kinase during CSF-1-induced FD/WT differentiation to macrophages (20). Nevertheless, the exact signaling mechanism(s) that are abrogated by the Y807F mutation and critical to differentiation are poorly understood (16).
In contrast to FD/807 cells where there is a defect in differentiation, fibroblasts transfected with the human CSF-1RY809F, the human equivalent of the Y807F mutation, were found to be incapable of proliferation (3). No differentiation phenotype exists for the CSF-1R-transfected fibroblasts, and the Y809F substitution did not modulate the accumulation of mRNA of two transcription factors, c-Fos and JunB (21). In other cell systems, modulation of c-fos mRNA levels has been associated with macrophage lineage differentiation (22,23) as well as with osteoclast differentiation (24). Taken together, these reports suggest that the murine Tyr 807 (or human Tyr 809 ) may be involved in signaling pathway(s) that, depending on the cell system, are involved in either differentiation and/or proliferation.
The p46/52 Shc adaptor protein is widely expressed and is composed of a C-terminal SH2 domain and an N-terminal protein tyrosine binding domain (25). Treatment of human monocytes with CSF-1 results in the tyrosine phosphorylation of p46/52 Shc and its association with Grb2 (12). A complex of Grb2 and p46/52 Shc was found to co-immunoprecipitate CSF-1R following treatment of FD/WT cells with CSF-1 (9). In other cell systems, p46/52 Shc also forms complexes with p125 FAK and PI 3-kinase (11,12). Given the recurring observation of Grb-2 and p46/52 Shc association in a range of cell types, it is interesting that the CSF-1R-mediated tyrosine phosphorylation of p46/52 Shc is unaffected by the Y697F substitution at the Grb2 binding site (9). This implies that there may be a CSF-1R site(s) involved in p46/52 Shc phosphorylation that has yet to be identified.
While the Tyr to Phe substitution at position 807 of the CSF-1R in FD cells leads to a loss of the ability to differentiate into macrophage-like cells in response to CSF-1, the molecular basis for this is yet to be determined. Examination of the tyrosine phosphorylation profile of Nonidet P-40 lysate proteins in CSF-1-treated FD/WT and FD/807 cells by one-dimensional SDS-PAGE has failed to reveal any differences (16). We used the higher resolving power of two-dimensional SDS-PAGE to examine the phosphotyrosine patterns of CSF-1treated FD/WT and FD/807 cells to identify tyrosine-phosphorylated proteins that may be involved in macrophage differentiation.
Cells-The murine hemopoietic FD/WT and FD/807 cell lines were obtained from Drs. K Carlberg and L. Rohrschneider (Department of Pathology, University of Washington, Seattle) and have been described previously (16).
M1 murine myeloid cells were transfected with wild type c-fms (M1/ WT) or c-fms with the tyrosine mutated to phenylalanine (M1/807). Briefly, wild type c-fms (obtained from D. Hume, University of Queensland, Brisbane, Australia) and a Y807F c-fms mutant were cloned into the pEF-BOS expression vector (26). Mutation of tyrosine 807 to phenylalanine was generated by PCR using mutagenic primers that substituted the codon of Y807F from TTC to TAC. All PCR fragments were generated using the Expand High Fidelity PCR system (Roche Molecular Biochemicals), and mutant clones were fully sequenced prior to experimental use. The PCR fragments containing the mutations were then cloned into the wild type c-fms gene. The expression vectors were introduced into M1 cells as described previously (27). The capacity of M1/WT cells to adopt a macrophage morphology in response to treatment with CSF-1 was examined as before (27) and confirmed to be similar to that of FD/WT cells (16). Similarly, the inability of M1/807 cells to adopt a macrophage morphology in response to treatment with CSF-1 was examined and found to be similar to that of FD/807 cells (16).
Murine DNA constructs encoding Myc-tagged forms of either wild type p52 Shc or a non-tyrosine-phosphorylatable form of p52 Shc with Tyr to Phe substitutions at positions 239, 240, and 317 were a kind gift from Dr. Benjamin Margolis (28). They were transfected using the Fu-GENE TM 6 transfection reagent (Roche Molecular Biochemicals) as per the manufacturer's instructions. Briefly, 3 l of FuGENE TM 6 transfection reagent was diluted in 100 l of serum-free DMEM for 10 min. To this, 1 g of cDNA was added to achieve a final concentration of 0.1 g/ml. After incubation at room temperature for 15 min, this mixture was added dropwise to 2 ml of M1/WT cells at 1 ϫ 10 5 /ml. Cells were then cultured for 14 days in DMEM with 10% (v/v) heat-inactivated new born calf serum containing puromycin (2 g/ml). Selected cells expressing either the wild type p52 Shc (M1/WT WTShc ) or a non-tyrosine-phosphorylatable form of p52 Shc with Tyr to Phe substitutions at positions 239, 240, and 317 (M1/WT F3Shc ) were isolated. Pooled transformants were used in all experiments.
Cell Culture-FD and M1 cell lines were grown, growth-arrested, and treated with either CSF-1 or IL-3 as described previously (16,27).
Western Blot Analysis of SDS-PAGE Gels-Gels were analyzed by Western blotting as before (29). In some cases the blots were stripped of antibodies by extensive washing in stripping buffer containing 62.5 mM Tris/HCl, 0.1 M ␤-mercaptoethanol, 2% SDS, blocked with a 3% (w/v) bovine serum albumin, 1% (w/v) ovalbumin solution for 1 h at room temperature, then re-probed with different antibodies as described elsewhere (30).
Immunoprecipitation-Lysates were precleared by incubating 500 g of Nonidet P-40 lysates, from appropriately treated cells, with 50 l of a 25% (v/v) slurry of protein A-Sepharose 4B (Amersham Pharmacia Biotech) with rotation for 30 min at 4°C. The samples were then centrifuged at 13,000 rpm for 5 min and the pellets discarded. They were then incubated with 1 g of 4G10 antibodies (Upstate Biotechnology, Inc.) overnight with rotation at 4°C. Fifty microliters of a 25% (v/v) slurry of protein A-Sepharose 4B was added and the mixture incubated for 1 h at 4°C. The beads were pelleted and washed three times with cell lysis buffer. Samples were boiled for 5 min in SDS-PAGE sample buffer, subjected to one-dimensional SDS-PAGE on 10% SDS-PAGE gels, and transferred to nitrocellulose for Western blot analysis. Protein Staining-Membranes were stained with Colloidal Gold Total Protein Stain (Bio-Rad). SDS-PAGE gels were stained overnight with 0.1% (w/v) Coomassie Blue (PhastGel Blue R350 Stain; Amersham Pharmacia Biotech) in 25% (v/v) methanol with 10% (v/v) acetic acid, destained in 25% (v/v) methanol with 10% (v/v) acetic acid, and dried.
Quantitative Determination of Phosphorylation-ECL autoradiographs were scanned using a computerized laser densitometer (Molecular Dynamics, CA) and the images analyzed using Image Quant software (Molecular Dynamics).

RESULTS
Proteomic Analysis of Tyrosine Phosphorylation in CSF-1stimulated FD/WT and FD/807 Cells-Nonidet P-40 lysates of unstimulated and CSF-1-stimulated (5 min) FD/WT and FD/ 807 cells were resolved by two-dimensional SDS-PAGE, trans-ferred to nitrocellulose, probed with biotinylated 4G10 antibodies, and developed with streptavidin-conjugated HRPO. Cursory examination of the two-dimensional SDS-PAGE Western blots revealed dramatic up-regulation of tyrosine phosphorylation of many proteins following CSF-1 addition (Fig. 1, panels A-C). Coomassie Blue staining was used as an indication of the consistency of the loading and resolution (Fig. 1, panels D and E).
Closer examination of the two-dimensional SDS-PAGE phosphoprotein profiles of FD/WT and FD/807 cells revealed both common and distinct tyrosine phosphorylation of proteins by CSF-1 (Fig. 1, panels A-C). Major phosphoproteins were classified on the basis of their molecular weights and pI. For example, a protein with a molecular mass of 74 kDa and a pI of 6.0 was designated 74/6.0 (Fig. 1, panels A-C). Each of the phosphoproteins or "spots" indicated in this fashion in Fig. 1 (panels A-C) was subjected to densitometric analysis and quantification (Fig. 2).
The CSF-1-induced tyrosine phosphorylated proteins were classified as belonging to one of two groups: those whose tyrosine phosphorylation was consistently "unaffected" by the Y807F substitution and those whose tyrosine phosphorylation was consistently "affected." These phosphorylation patterns are depicted qualitatively by a representative series of 4G10 antibody Western blots in Fig. 1, panels A-C. In each case, we have attempted to show as complete a two-dimensional SDS-PAGE gel as possible, focusing on the region of greatest interest, namely that containing the highest number of clearly identifiable affected and unaffected phosphoproteins. The average intensities, from five different experiments, are represented quantitatively in Fig. 2. As an example, spots 74/6.0, 67/7.8, 66/6.5, 64/5.8, 62/6.7, 62/5.9, 60/7.0, 27/7.4, and 27/6.7 belong to the unaffected group (Fig. 1, panels A-C; Fig. 2, panels A-I, respectively). The affected group was divided into those spots whose CSF-1-dependent phosphorylation state was "diminished" by the Y807F substitution or, alternatively, those whose CSF-1-dependent tyrosine phosphorylation was "elevated" by the Y807F substitution. The diminished group included tyrosine-phosphorylated proteins, 74/5.7, 66/6.1, 55/7.7, 52/6.9, 52/6.7, 46/6.3, 42/6.7, 37/6.7, 27/6.1, and 24/6.1 (Fig. 1, panels A-C; Fig. 2, panels J-S, respectively); the elevated group of molecules included the 100/8.5 and 100/8.0 (Fig. 1, panels A-C; Fig. 2, panels T and U, respectively). Since the tyrosine phosphorylation status of the affected group of proteins correlates with a change in the capacity of the FD cells to differentiate in the presence of CSF-1, it is possible that proteins in this group may play a role in the differentiation process.
Differential Tyrosine Phosphorylation of p46/52 Shc -The phosphorylation of spots 52/6.9, 52/6.7, and 46/6.3 was dramatically abrogated by the Y807F substitution (Figs. 1, panels A-C; Fig. 2, panels M-O, respectively). Their molecular weights suggested that these proteins might be p46/52 Shc ; however no such Y807F-dependent modulation in the tyrosine phosphorylation status of p46/52 Shc was reported before in CSF-1-treated FD cells (9). We, therefore, addressed this question further. The  Cells were growth-arrested in medium containing 1% serum for 18 h (unstimulated) and then treated with CSF-1 (5000 units/ml) for 5 min (ϩCSF-1). Nonidet P-40 lysates were standardized for protein and resolved by isoelectric focusing pH 3-10 and SDS-PAGE (see "Experimental Procedures"). Gels were either transferred to nitrocellulose and probed with 4G10 antibodies (panels A-C) or stained for protein loading with Coomassie blue (panels D-F). The location of phosphoproteins of interest (see "Results"), their pI and molecular masses are indicated with arrows.
two-dimensional SDS-PAGE blots were stripped and re-probed with anti-p46/52 Shc antibodies. The identification of the 52/6.9, 52/6.7, and 46/6.3 proteins by both anti-p46/52 Shc and 4G10 antibodies suggested that they were p46/52 Shc and that the CSF-1-dependent tyrosine phosphorylation evident in FD/WT cells was abrogated in FD/807 cells (Fig. 3). In addition to phosphoproteins 52/6.9, 52/6.7, and 46/6.3, which were all identified by p46/52 Shc -reactive antibodies, a number of p46/52 Shc proteins (Fig. 3, panels D--F) did not appear to co-migrate with CSF-1-sensitive phosphoproteins (Fig. 3, panel B), suggesting that not all the p46/52 Shc isoforms are phosphorylated. Similarly, the phosphorylation of the 52/6.9, 52/6.7, and 46/6.3 phosphoprotein p46/52 Shc isoforms would appear to alter their pI mobility, probably by the addition of charged phosphate species. It is interesting to note that the pI distribution of p46/ 52 Shc isoforms in unstimulated FD/WT and CSF-1-treated FD/ 807 cells are nearly identical, possibly corresponding to an "unphosphorylated" pattern and are different to that of CSF-1-treated FD/WT cells, which may represent a "phosphorylated" pattern. The identity of these phosphoproteins was further examined by amino acid sequence determination as described previously (31). Consistent with data presented above, phosphoproteins 52/6.9, 52/6.7, and 46/6.3 were found to be the p46/52 Shc adapter protein (data not shown).
Given this result, we sought to determine directly whether p52 Shc was tyrosine-phosphorylated to different degrees in FD/WT and FD/807 cells treated with CSF-1. Nonidet P-40 lysates from untreated and CSF-1-stimulated FD/WT and FD/ 807 cells were either immunoprecipitated with 4G10 or anti-p52 Shc antibodies, resolved by one-dimensional SDS-PAGE, transferred to nitrocellulose, and probed with the converse antibody, that is, either anti-p52 Shc or 4G10 (Fig. 4). Each approach revealed that the maximal CSF-1-stimulated tyrosine phosphorylation of p52 Shc protein was dependent on the tyrosine at position 807 (Fig. 4). To determine whether this observation was simply cell type-specific we repeated the above examination with another murine myeloid cell system, namely the M1/WT and differentiation-deficient M1/807 cells (see "Experimental Procedures"). Again, the Y807F mutation in the CSF-1R resulted in a dramatically reduced tyrosine phosphorylation of p52 Shc in response to CSF-1 treatment (Fig. 4). The combination of these approaches suggests that the Tyr at position 807 plays a part in p52 Shc tyrosine phosphorylation rather than simply in the association, direct or otherwise, with tyrosine-phosphorylated proteins.
As a check on the specificity of our observations for p52 Shc , we examined, for each cell type, the tyrosine phosphorylation of the p85 subunit of PI 3-kinase using the same approach and in this case found it to be CSF-1-dependent and Y807F mutationindependent (Fig. 4). These findings suggest that p52 Shc may have a role in CSF-1-mediated macrophage differentiation in both of these cell systems. (32)(33)(34)(35)(36)(37). The loss of p46/52 Shc binding to the epidermal growth factor receptor family member ErbB3 has been shown to result in the loss of ERK activation (38). Accordingly, given the loss of p52 Shc tyrosine phosphorylation following the Y807F substitution, we sought to examine the consequent effect of this mutation on ERK activity. The phospho-ERK immunoreactivity of M1/WT and M1/807 cells treated with CSF-1 for 4, 10, and 30 min was examined. The antibody binds specifically to the active forms of ERK and revealed that the Y807F mutation dramatically abrogated ERK activation (Fig. 5). Interestingly, the specific MEK inhibitor, PD98059, inhibits both CSF-1-induced ERK activation and differentiation of M1 cells. 2 Therefore, it could be that, in a fashion analogous to the ErbB3 system (35), ERK activation and differentiation are compromised through the loss of p52 Shc activation following the Y807F mutation.

The Y807F Substitution Prevents Maximal CSF-1-mediated ERK Activation-ERK family members can play a role in cellular differentiation
Expression of a Non-tyrosine-phosphorylatable Form of p46/ 52 Shc Prevents CSF-1-mediated Macrophage Differentiation-As the tyrosine phosphorylation of p46/52 Shc was abrogated by the Tyr to Phe substitution at position 807 of the CSF-1R, which also dramatically affects macrophage differentiation, we sought to determine whether the prevention of p46/52 Shc tyrosine phosphorylation could similarly abrogate macrophage differentiation. Myc-tagged forms of either wild type p52 Shc or a non-tyrosine-phosphorylatable form of p52 Shc with Tyr to Phe substitutions at positions 239, 240, and 317 were transfected into M1/WT cells to produce M1/WT WTShc or M1/WT F3Shc cells, respectively. Quiescent M1/WT WTShc and M1/WT F3Shc cells were either left untreated or stimulated with CSF-1 and Nonidet P-40 lysates prepared. These were subjected to Western blot analysis using p46/52 Shc -, Myc-, or phosphotyrosine-reactive antibodies following immunoprecipitation with either p46/52 Shc or Myc-reactive antibodies (Fig. 6). p46/ 52 Shc underwent CSF-1-induced tyrosine phosphorylation in M1/WT WTShc , but not M1/WT F3Shc , cells consistent with previous reports (Fig. 6; Ref. 28). Interestingly, the endogenous Cells were growth-arrested in medium containing 1% serum for 18 h (Ϫ) and then treated with CSF-1 (5000 units/ml) for 5 min (ϩ). Nonidet P-40 lysates were standardized for protein and immunoprecipitated with 4G10 antibodies, resolved by one-dimensional SDS-PAGE, transferred to nitrocellulose, and probed with either anti-p52 Shc or anti-PI 3-kinase p85 subunit antibodies. p52 Shc , immunoglobulin heavy chain, and the p85 subunit of PI 3-kinase are indicated with arrows.
p46 Shc and p52 Shc forms failed to be tyrosine-phosphorylated in M1/WT F3Shc cells in response to CSF-1 treatment, suggesting a dominant negative effect.
The functional significance of the loss of p46/52 Shc tyrosine phosphorylation in M1/807 and M1/WT F3Shc cells was next investigated. M1/WT, M1/807, M1/WT WTShc , and M1/WT F3Shc cells were cultured in DMEM containing either HINBCS alone or with CSF-1 for 4 days. Consistent with previous reports in analogous FDC-P1 cells transfected with the CSF-1R (16), the wild type CSF-1R permitted CSF-1-dependent macrophage differentiation in M1 cells as determined by cell spreading, adhesion, blebbing, and loss of blast-like morphology (Fig. 7A). Similarly, these cell lines were assessed by flow cytometry for their capacity to express the monocyte/macrophage lineage marker, Mac-1, in response to CSF-1 treatment (Fig. 7B). The observation that both M1/807 and M1/WT F3Shc cells were incapable of CSF-1-mediated differentiation (Fig. 7, A and B) suggests that the Tyr 807 of the CSF-1R-dependent macrophage differentiation process in this system is mediated through the tyrosine phosphorylation of p46/52 Shc .
The association of the CSF-1R with p46/52 Shc following specific ligand stimulation is well established (9, 10). We, therefore, sought to determine the effect of the F3 mutation of Shc, given the impairment of CSF-1-mediated macrophage differentiation, on the association of p46/52 Shc and the CSF-1R. p46/ 52 Shc was immunoprecipitated from CSF-1-treated M1/WT, M1/807, M1/WT WTShc , and M1/WT F3Shc cell lysates, resolved by one-dimensional SDS-PAGE, transferred to nitrocellulose, and probed with HRPO-conjugated 4G10 antibodies (Fig. 8, panel  A). p46/52 Shc was observed to be phosphorylated maximally at 1 and 5 min post-stimulation in M1/WT and M1/WT WTShc cells and this diminished progressively at 15 and 30 min, respectively, in a manner consistent with previous reports (39). This phosphorylation was dramatically reduced in M1/807 cells as described earlier (see Fig. 4). Interestingly, M1/WT F3Shc cells contained significantly higher levels of phosphorylated p46/ 52 Shc relative to M1/807 cells, presumably corresponding to endogenous p46/52 Shc , but this was dramatically reduced relative to that seen in M1/WT and M1/WT WTShc cells (Fig. 8, panel A).
Stripping and re-probing of the membrane depicted in Fig. 8, panel A, with anti-c-Fms antibodies was performed (Fig. 8,  panel B). The expected CSF-1-dependent association of p46/ 52 Shc and c-Fms was observed in M1/WT and M1/WT WTShc cells. Of significant interest was the reduced association of p46/52 Shc and c-Fms in both M1/807 and M1/WT F3Shc cells. This is, presumably, due to the inability of a nonphosphorylated p46/52 Shc to bind to either the CSF-1R or other proteins that are themselves associated with the CSF-1R. As M1/ WT F3Shc cells contain an otherwise normal CSF-1R, unlike that expressed in M1/807 cells, this lack of association is unrelated to the activation of the CSF-1R itself. DISCUSSION The molecular basis for Y807F-dependent loss of macrophage differentiation in FD/807 cells was examined. One-dimensional SDS-PAGE and Western blot analysis of tyrosine phosphorylated proteins in Nonidet P-40 lysates revealed an  (27). These experiments were repeated three times.
increased and largely conserved pattern of tyrosine-phosphorylated proteins in FD/WT and FD/807 cells following CSF-1 treatment (16). In contrast, a more detailed examination by two-dimensional SDS-PAGE revealed dramatic differences in the tyrosine phosphorylation patterns of FD/WT and FD/807 cells treated with CSF-1. A number of tyrosine-phosphorylated proteins were observed to be modulated by treatment with CSF-1, including the 52/6.9, 52/6.7, and 46/6.3 proteins that were also identified by p46/52 Shc -reactive antibodies and whose tyrosine phosphorylation was down-regulated by the Y807F mutation. Subsequent immunoprecipitation, Western blotting, and in vitro binding studies supported the view that the 52/6.9, 52/6.7, and 46/6.3 proteins were p46/52 Shc . The transfection of a non-tyrosine-phosphorylatable form of p52 Shc with Tyr to Phe substitutions at positions 239, 240, and 317 conferred a differentiation-incapable phenotype to M1/WT cells that closely reflected the differentiation-deficient behavior of M1/807 cells. These results are the first to suggest that p46/52 Shc , and more specifically its tyrosine phosphorylation, may play a role in macrophage differentiation.
These findings are in contrast to a previous report showing the tyrosine phosphorylation of p52 Shc was unaffected by the Y807F in FD cells (9). This prior study reported that immunoprecipitates of p52 Shc from FD/WT and FD/807 cells exhibited similar 4G10 antibody immunoreactivities (9). These discordant findings are difficult to reconcile. One possibility is that in the earlier study (9), the comparative analysis of p52 Shc phosphorylation in FD/WT and FD/807 cells did not include an unstimulated control group for either of these cells. Rather, parental FD cells were used, which cannot respond to CSF-1. As in our studies above, the inclusion of unstimulated FD/WT and FD/807 cells as controls may have revealed the effects of the Y807F mutation on the phosphorylation state of p52 Shc . In addition, we have used complementary and reverse combinations of Western blot and immunoprecipitation analysis with anti-p52 Shc and 4G10 antibodies to observe that p52 Shc tyrosine phosphorylation is diminished in FD/807 cells compared with FD/WT cells in response to CSF-1. As an added check on our findings, the analysis was repeated in another myeloid cell line, namely M1 cells, to exclude the possibility of a cell linespecific artifact. More importantly, the ability of a non-tyrosine-phosphorylatable form of p52 Shc to abrogate CSF-1-dependent monocyte/macrophage differentiation in otherwise competent M1/WT cells provides the strongest evidence that the tyrosine phosphorylation of p52 Shc is important in these processes.
There are other reports in growth factor/receptor systems that suggest a role for p46/52 Shc in cellular differentiation (37, 38, 40 -46). The c-Mpl ligand, MGDF (or TPO), induces basophil-like differentiation in 32D cells transfected with murine c-Mpl (37,38). This differentiation has been shown to involve p46/52 Shc phosphorylation (38). The Tyr to Phe substitution at position 559 on the c-Mpl receptor in WEHI3D-B ϩ cells abrogates both differentiation and p46/52 Shc phosphorylation (40), further suggesting a role for p46/52 Shc in hemopoietic lineage differentiation. Indeed, a mutant form of p46/52 Shc lacking a phosphotyrosine interaction domain blocks c-Mpl-mediated granulocytic differentiation (41). It has been shown in neuronal cells that differentiation signals are controlled by the NGF Receptor/Trk binding sites for p46/52 Shc and phospholipase C-␥ (41,43,44). It is interesting to note that the tyrosine phosphorylation of phospholipase C-␥2 in CSF-1-treated FD/807 cells is diminished (20) in a fashion that parallels the PC12 neuronal cell differentiation model (43). The Y577F substitution in the GM-CSF receptor in murine pro-B BA/F3 cells resulted in the loss of p52 Shc phosphorylation without affecting the activation of the p21 ras /Raf-1/MEK/MAPK cassette, the JAK/STAT pathway, and proliferation, suggesting that p52 Shc phosphorylation in not involved in proliferation (45).
The inability of FD/807 cells to differentiate in response to CSF-1 suggests that a molecular interaction critical to differentiation signaling is lost. Our work has revealed that p46/ 52 Shc may be part of the CSF-1-mediated differentiation signaling pathway. The mechanism by which the loss of p46/52 Shc activation may lead to an abrogation of differentiation remains unclear. A mutation of the p46/52 Shc binding site on epidermal growth factor receptor family member ErbB3 leads to the loss of heregulin activation of MAP kinases (35). The activation of ERKs is considered important in cellular differentiation signaling in a number of cell systems (32)(33)(34). For example, retinoic acid-induced myeloid differentiation of HL-60 cells involves the actions of MEK and ERKs (36). Of greater direct relevance is the report that ERK activation is impaired in Tyr 809 to Phe mutant forms of the CSF-1R-transfected NIH-3T3 cells (46). Consistent with these findings in other cell systems is our observation that differentiation-deficient FD/807 cells exhibit impaired CSF-1-dependent Erk1/2 activation, and this may be related to or caused by the loss of p52 Shc phosphorylation. FIG. 8. Analysis of the tyrosine phosphorylation state and association of p52 Shc and CSF-1R in Nonidet P-40 lysates of M1/WT, M1/807, M1/ WT WTShc , and M1/WT F3Shc cells treated with CSF-1. Cells were growtharrested in medium containing 1% serum for 18 h and then treated with CSF-1 (5000 units/ml) for 0, 1, 5, 15, and 30 min and Nonidet P-40 lysates prepared as described under "Experimental Procedures." Panel A, p46/52 Shc immunoprecipitates were resolved by 10% onedimensional SDS-PAGE gel, transferred to nitrocellulose, and probed with 4G10 antibodies directly conjugated with HRPO. p46/52 Shc is indicated with arrows. Panel B, the membrane described above was stripped and reprobed with anti-c-Fms antibodies. c-Fms and IgG are indicated with arrows.


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Characterization of a Novel Carotenoid Cleavage Dioxygenase from Plants*

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction,nine- c is epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9–10 and 9′-10′ positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C14 dialdehyde and two C13 products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9 -10 and 9-10 positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C 14 dialdehyde and two C 13 products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.
Apocarotenoids are a class of compounds derived from the oxidative cleavage of carotenoids (1). This is a structurally diverse class of compounds that are widely distributed in nature. The assortment of apocarotenoids results from the large number of carotenoid precursors (more than 600 carotenoids have been identified), variations in the site of cleavage, and modifications subsequent to cleavage. Abscisic acid (ABA) 1 , which is derived from the oxidative cleavage of a 9-cis-epoxycarotenoid, is necessary for seed development and adaptation to environmental stresses in plants (2). Although historically referred to as retinoids, vitamin A and related compounds are derived from the cleavage of ␤-carotene (3).
Plants produce a number of volatile apocarotenoids. These compounds probably serve a function as insect attractants and are valued as flavor/aroma compounds. Apocarotenoid pig-ments also have some economic value. For example, bixin (annatto) is commonly used as a natural food coloring, and crocin is the major pigment in saffron (4).
The formation of apocarotenoids may result from nonspecific mechanisms, such as lipoxygenase cooxidation or photooxidation. Enzymes capable of cleaving carotenoids at specific sites are believed to be involved in the synthesis of a number of apocarotenoids. The Vp14 gene, which encodes an ABA biosynthetic enzyme in maize (5,6), was the first carotenoid cleavage enzyme to be cloned from any organism. Since the characterization of VP14, ABA biosynthetic enzymes have been identified in several plants (7)(8)(9)(10). Based upon sequence similarity, an enzyme necessary for vitamin A biosynthesis has been identified in Drosophila (11) and vertebrates (12). Another carotenoid cleavage enzyme in animals that reacts with the 9 -10 double bond has also been identified (13). The definitive demonstration of both symmetric and asymmetric cleavage pathways appears to resolve a long standing debate concerning vitamin A biosynthesis (14).
A number of hypothetical proteins that are similar to these carotenoid cleavage enzymes have also been identified in the sequence data bases, but their biochemical functions have not yet been demonstrated. In the Arabidopsis genome sequence, nine potential carotenoid cleavage enzymes have been identified. Most of these genes have been annotated as neoxanthin cleavage enzymes or nine-cis-epoxy-carotenoid dioxygenases (NCEDs). Both terms imply an involvement in ABA biosynthesis. Although it is likely that two or more genes encode ninecis-epoxy-carotenoid dioxygenases, it is doubtful that all nine genes are involved in ABA biosynthesis. Some of these proteins may catalyze carotenoid cleavage reactions not involved in ABA biosynthesis. Others might catalyze a double bond cleavage reaction of substrates other than carotenoids, such as the lignostilbene dioxygenases from prokaryotes (15,16). In this paper, a putative carotenoid cleavage enzyme from Arabidopsis was expressed in Escherichia coli. It is shown that recombinant protein catalyzes a carotenoid cleavage reaction different from the one in ABA biosynthesis. The gene symbol CCD, Carotenoid Cleavage Dioxygenase, was adopted in this paper to distinguish this gene family from genes that encode ABA biosynthetic enzymes.

EXPERIMENTAL PROCEDURES
Constructs-A truncated cDNA for the AtCCD1 gene was obtained from the Arabidopsis expressed sequence tag collection (N95924). The full-length cDNA sequence (19) (AJ005813) and the genomic sequence (AL163818; gene identification MAA211 50) have also become available. A full-length clone was obtained by reverse transcription-polymerase chain reaction using RNA from light-grown seedlings. The polymerase chain reaction product was amplified with Pfu polymerase (Stratagene) and the following primer sequences: 5Ј-CATGGCGGAGAAACTCAGTG-3Ј and 5Ј-TTATATAAGAGTTTGTTCCTGG-3Ј. The amplified fragment was cloned into the SmaI site of pBluescript SK (Stratagene) and pGEX-2T (Amersham Pharmacia Biotech) to produce pCZY1 and pCZY2, respectively. A fragment of the PvCCD1 gene in beans, previously called PvNCED2, was amplified with degenerate primers as previously described (7). The flanking sequences were obtained by polymerase chain reaction from a bean cDNA library with one primer annealing to the initial polymerase chain reaction product and either a T7 or T3 primer annealing to the vector. Once the 5Ј and 3Ј sequences were determined, a full-length clone (AY029525) was amplified from the bean library with the following primer sequences: 5Ј TGGATCCATGGGGGATGATGG 3Јand 5Ј-TG-GATCCTCACAGTTTTGCTTG-3Ј. The BamHI restriction sites added on to the primer were used to clone into the BamHI site of pGEX-2T (Amersham Pharmacia Biotech) to create pXQ1.
Protein Expression and Enzyme Assays-A 5-ml culture of pCZY2 or pXQ1 was used to inoculate a 100-ml culture in 2ϫ YT medium (per liter: 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl). Cultures were grown at 37°C until an A 600 of 0.7 was reached. Expression of the protein was induced by the addition of 0.2 mM isopropyl ␤-D-thiogalactopyranoside, and the cultures were grown at 28°C for an additional 3-5 h. The E. coli cells were harvested by centrifugation, resuspended in Tris-buffered saline, and lysed in a French press. The recombinant protein was purified with glutathione S-transferase-agarose (Sigma) and then released by cleavage with thrombin for 7 h at 4°C. The carotenoid substrates were extracted and purified as previously described (17). Assays contained 0.1% Triton X-100, 0.5 mM FeSO4, 5 mM ascorbate, and the appropriate carotenoid substrate in 100 mM Bis Tris, pH 7.0. The assay products were partitioned into ethyl acetate and analyzed by HPLC or thin-layer chromatography as previously described (5). The mass spectra for C 27 compounds were obtained by direct inlet as previously described (5). For gas chromatography-mass spectrometry of the C 13 (18) were cotransformed into E. coli strain JM109 with the pCZY1 or pCZY2 constructs described above. An overnight culture of 2 ml was used to inoculate 50 ml of LB medium containing the appropriate antibiotics and 0.1 mM isopropyl ␤-D-thiogalactopyranoside. The cultures were grown at 30°C for 24 h. The E. coli culture was centrifuged, and the cell pellet was resuspended in an equal volume of formaldehyde. An equal volume of methanol was then added, followed by two volumes of ethyl acetate. The phases were separated by the addition of water, and the ethyl acetate phase was retained for HPLC analysis.
Following centrifugation, the medium from the cultures was partitioned twice with an equal volume of ethyl acetate. The ethyl acetate was evaporated under vacuum, and the extracts were saponified prior to HPLC analysis. The HPLC fractions were dried, resuspended in the appropriate solvent, and used for UV-visible or fluorescence spectroscopy. A rosafluene standard was prepared by the reduction of the C 14 dialdehyde with NaBH 4 .

RESULTS
In Vitro Characterization of AtCCD1-The AtCCD1 gene, originally called AtNCED1 (19) in Arabidopsis, encodes a protein with limited sequence similarity to the carotenoid cleavage enzymes in ABA biosynthesis (31% identical and 42% similar to VP14). Although it was suggested that the gene is involved in ABA biosynthesis (19), the biochemical function of the protein has not yet been reported. Hypothetical proteins that share much higher similarity to known ABA biosynthetic enzymes have since been identified in the Arabidopsis genome sequence.
A comparison of the AtCCD1 protein with hypothetical proteins from other plant species indicates that it is a member of a highly conserved subfamily of carotenoid cleavage-related proteins (Fig. 1). The AtCCD1 protein is 67% identical and 74% similar to a protein from avocados, PaNCED2 (9). A protein from beans, PvCCD1 (this paper), is 78% identical and 86% similar to AtCCD1. In addition, the expressed sequence tags FIG. 1. Alignment of the AtCCD1 protein with related sequences. PaNCED2 was previously identified in avocados (AF224670) (9); PvCCD1 (AYO29525) is an apparent ortholog of AtCCD1 from beans; AtNCED5 is a potential ortholog of VP14 that was identified in the Arabidopsis genome sequence (AC074176); AnaCCD1 is an open reading frame from the cyanobacterium Anabaena PCC 7120 (c307: sequence 1467-2894).

FIG. 2. Thin-layer chromatography analysis of assays with the recombinant AtCCD1 protein and various carotenoid substrates.
The substrates used were ␤,␤-carotene (␤-Car), lutein (Lut), zeaxanthin (Zeax), all-trans-violaxanthin (tViol), 9-cis-violaxanthin (9cViol), and 9Ј-cis-neoxanthin (9cNeox). Enzyme assay products were separated on a thin-layer silica plate that was developed in hexane, ethyl acetate, and 2-propanol (70:20:10). Following chromatography, the plate was sprayed with 2,4-dinitrophenylhydrazine to detect aldehydes and ketones. The products were also characterized by a combination of HPLC, UV-visible spectroscopy, and mass spectrometry. Several of the characterized products are labeled on the chromatogram (see Fig. 3). Residual substrates are indicated by asterisks. from a variety of plants share a high degree of similarity to AtCCD1 at the nucleotide and the deduced amino acid level. Based upon limited sequence data, genes with greater than 80% nucleotide identity are present in the expressed sequence tags of numerous plant species. A hypothetical protein from Anabaena PCC 7120, AnaCCD1, also shares some sequence similarity with AtCCD1 (38% identity and 48% similarity).
To determine the potential function of AtCCD1, the gene was cloned into a glutathione S-transferase fusion vector for expression in E. coli, and the recombinant protein was assayed for cleavage activity with a variety of carotenoid substrates. The assay products were separated by thin-layer chromatography and sprayed with 2,4-dinitrophenylhydrazine to visualize aldehyde and ketone products (Fig. 2). To generate the reaction scheme (Fig. 3), the assay products were also characterized by a combination of HPLC, UV-visible spectroscopy, and mass spectrometry. A variety of C 13 products, resulting from cleavage at the 9 -10 and the 9Ј-10Ј positions, were identified (e.g. II, V, and VI). In assays containing lutein, zeaxanthin, and alltrans-violaxanthin (Fig. 2, lanes 2, 3, and 4, respectively), the products did not separate from the carotenoid substrates by TLC. It was, however, possible to isolate these products by HPLC for further characterization. The red spot (I) in Fig. 2, lanes 1-6 was the C 14 dialdehyde resulting from symmetrical cleavage at the 9 -10 and 9Ј-10Ј positions (Fig. 3). This C 14 dialdehyde was a major product when ␤-carotene, lutein, zeaxanthin, or all-trans-violaxanthin was used as a substrate. The enzyme did not cleave as well adjacent to a 9-cis double bond or an allenic bond found in some carotenoids. In assays containing the 9-cis isomer of violaxanthin (Fig. 2, lane 5), the major product was the C 27 epoxy-apocarotenal (III) that results from a single cleavage distal to the cis double bond. When 9Ј-cisneoxanthin was used as a substrate (Fig. 2, lane 6), both a C 27 epoxy-apocarotenal (III) and a C 27 allenic-apocarotenal (IV) were produced.
FIG. 4. E. coli strains engineered to accumulate zeaxanthin and coexpressing CCD1 proteins. "AtCCD1" is a strain coexpressing pCZY1, and "PvCCD1" is a strain coexpressing pXQ1. The control strain contains the pGEX-2T vector with no insert. natural product of roses, which would be produced by the reduction of the C 14 dialdehyde to the corresponding dialcohol (20). Reduction of the C 14 dialdehyde with NaBH 4 did produce a compound with the same retention time and absorption spectrum (data not shown). Peak 2, which has a spectrum shifted to a slightly lower wavelength, is probably a cis isomer.
A gene sharing high sequence similarity to AtCCD1 was cloned from Phaseolus vulgaris and named PvCCD1, previously PvNCED2 (7). The PvCCD1 protein was also expressed in E. coli (Fig. 4) and shown to catalyze the symmetric cleavage of zeaxanthin to form the C 14 dialdehyde. DISCUSSION The recombinant AtCCD1 protein cleaves a variety of carotenoids at the 9 -10 and 9Ј-10Ј double bonds. With most substrates tested, the carotenoid is symmetrically cleaved to produce a C 14 dialdehyde. The prevalence of the C 14 product (symmetric cleavage) relative to the C 27 product (single cleavage) may indicate that the enzyme functions as a dimer, simultaneously cleaving at both ends. The lignostilbene dioxygenases from prokaryotes, which share sequence similarity to the carotenoid cleavage enzymes, are known to function as dimers (16). In the case of 9-cis-violaxanthin and 9Ј-cis-neoxanthin, the C 27 apocarotenals are major products. Cleavage adjacent to a cis double bond or the allenic bond of neoxanthin appears to be impaired.
The structural variations in carotenoids exist mainly in the rings. Because AtCCD1 is able to cleave a variety of carotenoids, a number of interesting C 13 ring products are generated in the in vitro assays. The grasshopper ketone (V), derived from the cleavage of neoxanthin, is regurgitated by some species of grasshoppers (21,22). The unpalatable taste of this compound may deter ants and other predators. Other C 13 compounds are valued as flavor and aroma constituents of a variety of foods (23). Some C 13 cleavage products are effective insect lures (24,25) and may serve as insect attractants when produced by plants.
Probable orthologs of the AtCCD1 gene are present in a wide range of plants. An ortholog in beans is able to catalyze the same reaction. The substrate of these enzymes in planta and the function of the apocarotenoid products are not yet known. However, a number of apocarotenoids have been identified in plants that are derived by an AtCCD1-like activity. The C 14 dialdehyde is the probable precursor of rosafluene, a highly fluorescent compound found in some varieties of roses (26). The C 14 product is also the expected precursor of mycorradicin, a yellow pigment that accumulates in the roots of plants inoculated with arbuscular mycorrhizal fungi (27). Several C 13 cleavage products are also abundant in the roots of infected plants.
Although Arabidopsis does not appear to form mycorrhizal associations, an AtCCD1-like protein probably catalyzes the synthesis of these apocarotenoids in other plant species.
A C 13 product of zeaxanthin or lutein cleavage, 3-hydroxy-␤ionone, accumulates in etiolated bean seedlings on exposure to light, and it has been suggested that this compound may have a function in the light-induced inhibition of hypocotyl elongation (28,29). The AtCCD1 gene is also induced by light in etiolated seedlings. 2 With the characterization of the AtCCD1 activity in vitro, it is now possible to speculate on the possible function of this gene in plants. By altering the expression of this gene in transgenic plants, it should be possible to characterize the apocarotenoid products and determine their biological role(s).


s2fieldsofstudy: Biology Medicine

text:
Xenobiotic acceleration of idiopathic systemic autoimmunity in lupus-prone bxsb mice.

The diverse genetic backgrounds of lupus-prone murine models, which produce both quantitative and qualitative differences in disease expression, may be a valuable resource for studying the influence of environmental exposure on autoimmune disease in sensitive populations. We tested this premise by exposing autoimmune-prone BXSB and the nonautoimmune C57BL/6 mice to the heavy metal mercury. Although both strains express a nonsusceptible H-2 haplotype, exposure to mercury accelerated systemic autoimmunity in both male and female BXSB mice, whereas the C57BL/6 mice were resistant. The subclasses of antichromatin antibodies elicited in BXSB mice by mercury exposure were more consistent with the predominant Th1-type response of idiopathic disease than with the Th2-type response found in mercury-induced autoimmunity (HgIA). The appearance and magnitude of both humoral and cellular features of systemic autoimmunity correlated with the mercury dose. Furthermore, environmentally relevant tissue levels of mercury were associated with exacerbated systemic autoimmunity. These studies demonstrate that xenobiotic exposure can accelerate spontaneous systemic autoimmunity, and they support the possibility that low-level xenobiotic exposure enhances susceptibility to systemic autoimmunity in genetically susceptible individuals.

Autoimmune diseases are associated with numerous immunologic and pathologic abnormalities, but the factors critical for inducing disease are poorly understood (1,2). Although genetic predisposition may be a prerequisite for the development of spontaneous systemic autoimmune diseases, such as systemic lupus erythematosus (SLE) (3), the incomplete concordance in SLE twin studies (4) suggests that exogenous or environmental factors are also important. Some of the best examples of systemic autoimmune disease triggered by environmental agents come from chemical-induced autoimmunity, and have been described both in humans (5,6) and in animal models (5,7,8). In addition, studies using murine models of lupus have shown that a variety of other exogenous agents can accelerate the onset of autoimmunity in genetically predisposed hosts (9)(10)(11)(12); however, no specific factor has been documented to play a vital role in idiopathic SLE.
Delineation of the role that environmental agents play in accelerating and/or exacerbating human autoimmune disease generally has not considered the fact that a subset of individuals may be particularly sensitive because of genetic predisposition. Although associations between environmental exposure and certain autoimmune diseases have been identified in epidemiologic studies (6,13), these studies have not identified those at risk. In addition, the genes that predispose to autoimmunity and their responses to environmental exposures have yet to be determined. Therefore, it has not been possible to test in humans whether genetic predisposition to spontaneous autoimmunity increases the sensitivity to environmental agents. The availability of animal models that spontaneously develop systemic autoimmunity provides an alternative approach to studying this question. Moreover, due to derivation from different genetic backgrounds, murine models of SLE manifest both quantitative and qualitative differences in disease expression that can be exploited (14,15).
Certain heavy metals, such as mercury, are potent environmental agents toxic to the immune system which can provoke not only immunosuppressive but also immunostimulatory effects in many species, including humans and rodents (reviewed in 8 and 16). Studies with nonautoimmune-prone animal models suggest that the immunoactivating properties of mercury can be divided into three major pathologic sequelae: lymphoproliferation, hypergammaglobulinemia, and the development of systemic autoimmunity manifested as production of autoantibody and immune-complex disease (8,15). Elicitation of these pathologic features depends on genetic background, with lymphadenopathy occurring in most strains, whereas autoimmunity-which in nonautoimmune-prone strains is controlled largely by the MHC gene-is more restricted (17,18). Exposure to mercury of autoimmune-prone MRL-+/+ and NZBWF1 mice accelerates autoimmunity in a strainspecific manner, with the most severe manifestations occurring in the NZBWF1 (19). Contrastingly, MRL-lpr mice, which are deficient for the Fas apoptosis-promoting gene and manifest more severe disease, exhibited little acceleration of humoral autoimmunity, suggesting that strains with highly accelerated disease may be less sensitive to environmental exposure. In addition, comparison with the H-2 compatible nonautoimmune-prone AKR mice indicated that both MHC and non-MHC genes contributed to acceleration of disease expression in the MRL-+/+. In these studies, lupusprone strains expressed MHC haplotypes that predispose to HgIA. Therefore, the extent to which susceptibility was due to HgIA or to underlying non-MHC susceptibility genes could not be clearly delineated.
In this study, we examined the influence of mercury exposure and dosage on expression of autoimmunity in the lupus-prone BXSB mice. In this strain, severe accelerated autoimmunity with early mortality occurs in males due to the Y-chromosome linked gene Yaa, whereas females develop a delayed and much milder form of disease (3,15). Importantly, the MHC haplotype of this strain (H-2 b ) is considered resistant to HgCl 2 since other strains with this haplotype, including the C57BL/6, which was used as the control in this study, are much less susceptible to HgIA than mice of otherwise similar backgrounds expressing s, k, or d haplotypes (8,17,18). Mercury exposure accelerated autoimmunity in BXSB mice consistent with idiopathic rather than HgIA disease based on the predominant types of IgG autoantibody subclasses detected. Dose-response studies suggested that environmentally relevant tissue levels of mercury were able to exacerbate systemic autoimmunity. These studies support the concept that low-level xenobiotic exposure can accelerate idiopathic systemic autoimmunity in genetically susceptible hosts.

Materials and Methods
Mice. Male and female BXSB (H-2 b ), and female C57BL/6 (H-2 b ) mice were obtained from The Scripps Research Institute Animal Colony (La Jolla, CA) and maintained under specific pathogen-free conditions. All experimental procedures using animals followed the guidelines set down in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996).
Treatment of mice. In short-term exposure studies, groups of up to eight 4-weekold mice were injected subcutaneously (sc) twice per week for 4 weeks with 100 µL PBS containing 40 µg HgCl 2 , or PBS alone. Mice were bled for sera before the first injection and at sacrifice on day 30. Autopsies were performed as described previously (20). Samples of kidney and spleen were taken for analysis of immune-complex deposition as described previously (see below).
Separate experiments were performed to examine for the persistence of autoimmunity following mercury exposure. Eight-week-old female BXSB and C57BL/6 mice were treated for 4 weeks with either HgCl 2 or PBS and then bled at 2-week intervals to assess autoantibody levels. At sacrifice, samples of kidney and spleen were obtained and proteinuria was determined.
To examine the effect of HgCl 2 dose on acceleration of autoimmunity, we injected groups of 8-week-old female BXSB mice with 40, 4, 0.4, or 0.04 µg HgCl 2 in PBS twice per week for 11 months. Control mice received PBS. Mice were monitored for disease as above. Samples of kidney were also analyzed for mercury content as described previously (21).
Serum immunoglobulin quantitation. Serum IgG, IgG1, and IgG2a levels were quantified by ELISA (26,27). ELISA plates were coated with 200 µL 2 µg/mL goat antimouse kappa light chain antibody (Caltag Laboratories) diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Plates were postcoated for 1 hr with 0.1% gelatin in PBS followed by 3 washes with PBS-0.05% Tween 20. Sera were diluted in serum diluent (26). A standard curve was generated by serial dilutions of polyclonal mouse reference serum containing predetermined levels of Ig isotypes (The Binding Site, Birmingham, UK). Diluted sera were incubated in duplicate while shaking for 2.5 hr followed by 3 washes with PBS-0.05% Tween 20. HRP-conjugated goat antimouse IgG, or IgG1 antibodies (Caltag Laboratories) were diluted in anti-Ig diluent (26) and incubated with shaking for 90 min. After 3 washes with PBS-0.05% Tween 20 and 4 washes with PBS, ABTS substrate solution was added and the optical density (OD) read at 405 nm. Determination of IgG2a in sera from BXSB and C57BL/6 mice required use of reagents specific for the b allotype of IgG2a (27). Serum IgG, IgG1, and IgG2a concentrations were calculated by extrapolation from the linear portion of standard curves.
Tissue immune-complex deposits. A 2-3 mm thick section of the kidney and spleen were snap-frozen in isopentane-CO 2 and examined by direct immunofluorescence as described previously (28). Briefly, 4-5 µm thick cryostat sections were fixed in ethanol and incubated with doubling dilutions of FITC-conjugated goat antibodies to IgG (gamma chain specific) and C3 (Southern Biotechnology Associates). The end-point titer of the immune deposits was defined as the highest dilution of antibody at which specific fluorescence could be detected. The presence of granular deposits in small and medium-sized arteries was also examined. The slides were examined under blinded conditions. Light microscopy. A 2-3 mm thick section of the kidney was immersed in Histochoice (Amresco, Solon, OH), and embedded in paraplast, and 1-2 µm sections were cut. The sections were stained with periodic acid-Schiff (PAS) reagent and with periodic-acid-silver-methenamine. The types of glomerular pathology were determined, and the degree of endocapillary cell hyperplasia was scored for each animal as follows: 0 = normal; 0.5 = just detectable alteration; 1 = slight; 2 = moderate; 3 = strong; and 4 = maximal. Slides were examined without knowledge of treatment or other results.
Urinary protein. Proteinuria was measured by Chemstrip 2 GP test strips as described by the manufacturer (Boehringer Mannheim Diagnostics, Indianapolis, IN). To compare results between groups, the milligram protein per deciliter scale was graded (0 = negative, 1 = trace, 2 = 30, 3 = 100, 4 = 500 mg/dL). Intermediate values were graded at 0.5 units above the lower value (i.e., 1.5 = trace to 30 mg/dL). In the mercury dose-response study, proteinuria was measured using the Bradford assay (Pierce, Rockford, IL) with BSA as the protein standard.
Statistical analysis. Unless otherwise noted, all data are expressed as mean ± 1SD. Groups were compared by unpaired t-test, single-factor analysis of variance, Mann-Whitney U test, or Fisher's Exact Test as appropriate. Comparisons are of HgCl 2treated mice with PBS-treated animals; p < 0.05 was considered significant.

Effects of short-term mercury exposure on 4-
week-old male BXSB mice. Although male BXSB mice are highly susceptible to SLE, 4 weeks of mercury exposure significantly elevated levels of serum IgG and IgG1 and IgG2a subclasses, compared to PBS control animals (Table 1). Antibodies to nuclear antigens (ANA) consisting of a dense fine to Articles • Pollard et al.

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VOLUME 109 | NUMBER 1 | January 2001 • Environmental Health Perspectives homogeneous nuclear speckling of interphase cells and metaphase chromosomes was found in 88% of male mice exposed to HgCl 2 , while pretreatment bleeds as well as PBStreated mice had less frequent ANA responses (range 0-25%). Similarly, levels of antichromatin antibodies in HgCl 2 -exposed male mice were elevated above those found in the PBS group (Table 1).
When compared to PBS-treated animals, HgCl 2 -treated male BXSB mice had increased organ wet weight for spleen and the draining cervical lymph nodes but not the mesenteric lymph nodes ( Table 2). Glomerular deposits, localized to the mesangium, were observed in both HgCl 2 -exposed and PBS-control animals, with mean titers higher in the mercury group (Table 2). This, however, did not reach statistical significance because of large variations in individual titers. Nevertheless, compared to PBS controls, mercury-exposed male BXSB mice showed significant increases in endocapillary cells as observed by light microscopy (1.62 ± 0.44 vs. 0.56 ± 0.050; p < 0.01) ( Figure 1). Glomerular basement membranes were normal and there was no inflammation. Interestingly, although nonglomerular deposits to the kidney vessel wall are typically seen in HgIA, they were absent in the mercury-exposed male BXSB mice. This suggests that the systemic autoimmunity induced in male BXSB mice by HgCl 2 was more consistent with spontaneous lupus than with HgIA.
Effects of short-term mercury exposure on 4-week-old female BXSB mice. Female BXSB mice are considerably less susceptible to SLE than their male counterparts, and develop mild SLE in late life. Nonetheless, mercury-exposed female BXSB mice also had elevations of serum IgG, IgG1, and IgG2a compared to PBS controls, although levels were lower than those in male HgCl 2treated mice (Table 1). Similar to male mice, 88% of females exposed to HgCl 2 developed ANAs that consisted of dense fine to homogeneous nuclear speckling of interphase cells and metaphase chromosomes (Table 1). PBS-treated mice, as well as all pretreatment bleeds, had less frequent ANA responses (range 0-29%). Although two HgCl 2exposed female mice had elevated antichromatin antibodies, the response of this group was not statistically different from the PBStreated group (Table 1).
HgCl 2 -treated female BXSB mice had greater organ wet weight for spleen and cervical lymph nodes but not mesenteric lymph nodes, compared to PBS treated animals ( Table 2). The two HgCl 2 -treated female mice with elevated antichromatin antibodies also had low titers of IgG deposits in the mesangium, whereas the remaining six HgCl 2 -treated mice as well as all mice given PBS showed no IgG deposits ( Table 2). The titers of glomerular C3 deposits were similar between the two groups. Histologic examination revealed increased glomerular endocapillary cells in HgCl 2 -treated mice (2.38 ± 0.92 vs. 1.29 ± 0.57, p < 0.05), but basement membranes were normal and there was no inflammation. Vessel wall deposits were not found in the kidney or spleen in either group.
Effect of short-term mercury exposure on nonautoimmune-prone 4-week-old female C57BL/6 mice. Compared to PBS-treated mice, mercury-exposed C57BL/6 mice developed hypergammaglobulinemia with   elevations in IgG, IgG1, and IgG2a and a dense fine speckled ANA pattern (Table 3). However, the mean antichromatin antibody level was not elevated (Table 3). One PBStreated mouse had an elevated antichromatin response (OD405 = 3.1), suggesting a low penetrance susceptibility to antichromatin autoantibody production in this strain. None of the mercury-treated C57BL/6 mice had increased deposition of immunoglobulin or complement (C3) in the kidney or spleen, and histology by light microscopy was unremarkable (data not shown). The lack of elevated antichromatin antibodies and kidney pathology in mercury-exposed C57BL/6 mice indicates that non-MHC genes are the major contributors to the HgCl 2 -induced responses in BXSB mice. Transient HgCl 2 exposure in 8-week-old female BXSB mice. To study the long-term effects of HgCl 2 following transient exposure, we gave 8-week-old female BXSB mice HgCl 2 or PBS for 1 month and then followed them for 32 weeks without further treatment. Mice were tested for autoantibodies at 2-week intervals and examined for immunopathology at the end of the experiment. C57BL/6 mice were treated similarly and tested only for autoantibodies because previous studies have already established that immunohistologic abnormalities do not develop in this strain [see above (17,18)].
The antichromatin response in HgCl 2treated BXSB mice was significantly elevated above that of PBS-treated mice up to 14 weeks after treatment (Figure 2), following which PBS-treated mice began to develop antichromatin antibodies. In contrast, HgCl 2treated C57BL/6 mice had low levels of antichromatin Ab, not significantly different from their PBS controls (data not shown). Compared to 4-week-old female BXSB (Table 1), older 8-week-old female mice were more susceptible to HgCl 2 -induced acceleration of antichromatin antibodies.
Histologic examination of kidneys revealed titers of glomerular IgG and C3 deposits that were higher in HgCl 2 -treated mice but varied widely among individual animals ( Table 4). Three of the control mice showed a combination of granular capillary wall and mesangial IgG staining, whereas the remaining four had only mesangial staining. Of the HgCl 2 -treated mice, one showed a combined granular capillary wall and mesangial IgG staining, two showed only granular capillary wall staining, and the remaining five mice had only mesangial staining. Mice with staining of the capillary loops, with or without concomitant mesangial staining, showed endocapillary cell hyperplasia. Most animals also had a moderate thickening and irregularity of the glomerular basement membrane. Mesangial deposits were associated only with slight endocapillary cell hyperplasia. Four of the HgCl 2 -treated but none of the PBS-treated mice had deposits of IgG in kidney vessels. The titer and frequency of immunoglobulin and complement deposits in the spleen was also higher in HgCl 2treated mice, with seven of eight having IgG deposits and six of those seven having C3 deposits, whereas only three PBS mice had low titer deposits of IgG and C3 in splenic vessels. The increased titers and frequency of immune reactants in the tissues of HgCl 2treated mice pointed to the development of more severe disease in these animals compared to PBS-treated mice. Consistent with this was the finding that three of the seven HgCl 2 -treated mice tested had > 30 mg/dL protein in their urine, whereas all five PBS mice tested had ≤ 30 mg/dL (p < 0.05; Table 4). Thus, compared to PBS controls,      HgCl 2 -treated mice have more severe disease long after termination of exposure.

IgG subclasses of antichromatin antibodies in BXSB mice with idiopathic and xenobiotic accelerated autoimmunity.
HgIA is associated with a Th2-like response characterized by increases predominantly in autoantibodies of the IgG1 subclass (30). HgCl 2 also has been shown to promote deviations from Th1 to Th2 predominance in several autoimmune diseases (27). In contrast, the spontaneous antichromatin response in BXSB mice is mainly of the IgG2a and IgG2b subclasses (31) due to the predominant Th1-like cytokine response in this strain (32). Thus, the IgG isotype autoAb levels of mercury-treated BXSB mice might indicate whether the accelerated autoimmunity resulted from the spontaneous (Th1) or HgIA (Th2) types of response. Sera from the long-term study of female BXSB mice treated transiently with HgCl 2 were examined for antichromatin IgG subclass levels. Immediately after 4 weeks of mercury exposure, all five antichromatin antibody-positive mice had subclasses that were predominantly of the IgG2a and IgG2b isotypes (Figure 3). Except for an IgG1 response in one mouse (No. 16), there were few antichromatin antibodies of the IgG1 and IgG3 subclasses. Thirty-two weeks after treatment began, the antichromatin IgG subclasses in PBS-and HgCl 2 -treated mice were very similar, consisting in particular of IgG2b as well as IgG2a subclasses. One PBS mouse (No. 3) had a strong IgG3 response, while HgCl 2 -treated mouse No. 16 had retained its IgG1 and IgG2a responses and increased its IgG2b response (Figure 3).

Effects of long-term exposure and lower doses of HgCl 2 on the development of autoimmunity in female BXSB mice.
Although the dose of HgCl 2 used in the above studies could accelerate autoimmunity in BXSB mice, the resulting tissue levels of mercury would be greater than that found in humans exposed to environmental levels of mercury (21,33,34). To determine whether lower levels of HgCl 2 exposure could also trigger autoimmune disease in female BXSB mice, we gave groups of 8-week-old animals either 40, 4, 0.4, or 0.04 µg HgCl 2 twice per week for almost 11 months. For mice given the highest dose of 40 µg, all succumbed or were moribund by 24 weeks of treatment after becoming severely cachexic. One mouse each in the 4 µg and 0.4 µg groups died at 31 and 38 weeks, respectively. Survival of mice in the 0.04 µg and PBS groups was not affected by treatment.
The antichromatin Ab response of female BXSB mice was clearly induced by exposure to HgCl 2 in a dose-dependent manner even with doses as low as 0.4 µg. Elevated levels were detected first in the 40 µg HgCl 2 group as soon as 4 weeks after the start of treatment and then 4 weeks later in the 4 µg HgCl 2 group (Figure 4). After 16-20 weeks of treatment, the 0.4 µg HgCl 2 group developed high levels of antichromatin Ab, equivalent to those in the 40 and 4 µg HgCl 2 groups. Antichromatin Ab levels subsequently remained elevated in these three groups for the remainder of the experiment. Even the lowest 0.04 µg HgCl 2 group tended to have higher antichromatin Ab levels than the PBS control at the end of the treatment period, but the levels did not reach those of groups given higher doses.
The accelerated autoimmune disease in male BXSB mice is associated with increases in memory/effector phenotype T cells and Mac-1 positive macrophages in the peripheral blood that may play a role in disease pathogenesis (14). When peripheral blood cells were examined after 32 weeks of HgCl 2 exposure, no changes were found in the percentage of peripheral blood mononuclear cell (PBMC) CD4 + T cells or B (B220 + ) cells (Table 5). However, mice receiving 4 µg HgCl 2 had increases in activated phenotype (CD44 hi ) CD4 + T cells (p < 0.005) and an increase in percentage of CD62L (Mel-14) lo CD4 + T cells, particularly when compared to the 0.04 µg HgCl 2 group (p < 0.025). Mice receiving 4 µg HgCl 2 also had an increased percentage of Mac-1 (CD11b) positive cells (p < 0.02). These increases in memory/effector and Mac-1 positive cells were less pronounced than that typically observed in male BXSB mice, and these specific manifestations may depend largely on the presence of the Yaa gene. Mice receiving 0.4 µg HgCl 2 had no statistically significant increases in PBMC phenotypic markers (Table 5).
We also examined urinary protein after 32 weeks of treatment. Mice in the 4 µg and 0.4 µg groups had increased proteinuria compared with PBS controls (p < 0.05), whereas mice receiving 0.04 µg HgCl 2 had similar levels (Table 5). After 38 weeks of treatment, the surviving mice were sacrificed and kidney pathology and mercury levels were examined. Mice in the 4 µg group had the most severe glomerular changes (average score ± SE, 2.2 ± 0.6); this was the only group that differed significantly from the PBS controls (score 0.6 ± 0.1, p = 0.03; Figure 5). The two mice from the 40 ug group that were sacrificed at 20 and 23 weeks had only mild changes (score 1.3 ± 0.3), suggesting that the early mortality from mercury toxicity was probably unrelated to glomerulonephritis. Interestingly, this was the only group that developed vascular deposits of IgG, a finding characteristically observed with HgIA. Mice in the 0.4 and 0.04 µg groups had glomerular changes similar to those in the PBS group (scores of 0.8 and 0.6, respectively). Mercury levels in the kidney (Table 6), which reflects the overall exposure of the animal, were significantly elevated for each of the doses compared to the PBS controls (p < 0.0001). There was a Articles • Xenobiotic acceleration of idiopathic autoimmunity Environmental Health Perspectives • VOLUME 109 | NUMBER 1 | January 2001  strong positive correlation between HgCl 2 dosage and kidney mercury levels (R = 0.99).

Discussion
In the present study, exposure of BXSB mice to mercury was shown to induce accelerated systemic autoimmunity in both the highly susceptible male and less susceptible female. Although we (19) and others (35) have shown that HgCl 2 exposure can induce systemic autoimmunity in other lupus-prone mice, in each instance the strains tested were susceptible to HgIA by virtue of their H-2 haplotypes alone. The current findings are significant in that the H-2 b haplotype of the BXSB does not predispose to HgIA (17,18). Therefore, induction of systemic autoimmunity by HgCl 2 exposure clearly implicates other susceptibility genes, most likely those related to spontaneous lupus. This study also demonstrates that exposure of BXSB mice to either a short course of high-dose mercury or lower doses over a long period could trigger systemic autoimmunity. For female BXSB mice, both the age of initial exposure and the dose of mercury were shown to influence the degree of disease exacerbation, particularly for antichromatin antibodies, an autoantibody closely associated with murine and human lupus (31,36). Moreover, a transient one-month exposure to mercury could elicit autoimmunity that persisted for many weeks after cessation of the xenobiotic and that had several features consistent with idiopathic disease.
Genetic studies show that both MHC and non-MHC genes can contribute to the expression of idiopathic systemic autoimmunity (14). In a previous study (19), comparison of the responses of MHC identical MRL-+/+ and AKR mice to mercury suggested that both MHC and non-MHC genes may contribute significantly to mercury toxicity. However, mercury exposure did not exacerbate humoral autoimmunity in the MRL-lpr mice, suggesting that HgCl 2 has little effect on mice that are already highly susceptible, or that it acts through the same anti-apoptotic mechanism as the Fas lpr mutation (19). In contrast, the present study found that the highly susceptible Yaa-expressing male BXSB mice were more sensitive to the autoimmune-promoting effects of HgCl 2 than female BXSB mice, which indicates that the addition of a strong autoimmune accelerating gene per se does not preclude xenobiotic acceleration of autoimmunity. Taken together with the MRL-Fas lpr results, this favors the possibility that HgCl 2 may promote HgIA by inhibiting the Fasinduced cell death of autoreactive cells. This is supported by recent evidence that HgCl 2 can protect against Fas-mediated cell death (37). Because mercury's effects are thought to be due primarily to reactivity to sulfhydryl groups, for which it has the highest binding (38,39), it is possible that mercury could be inhibiting cysteine proteases involved in the downstream events of Fas (40,41). Indeed, mutations of caspase 10 is one cause of the autoimmune lymphoproliferation syndrome in humans, a disease resulting from defective Fas-mediated apoptosis (42).
The accelerated autoimmunity in BXSB mice within the context of an MHC resistant to HgIA (43) suggests that autoimmunity in HgCl 2 -exposed lupus-prone mice is due to acceleration of idiopathic disease and not elicitation of HgIA. In addition, the predominance of IgG2a and IgG2b subclasses in the antichromatin Ab response supports stimulation of a Th1-like cytokine response, which is more consistent with spontaneous disease in the BXSB (32). Although gene knockout studies have revealed that HgIA is dependent upon interferon-γ (27), disease expression in nonautoimmune-prone wildtype mice is associated with a Th2 response with elevations in IL-4 and autoantibodies of IgG1 subclass (27,30). The lack of IgG1 antichromatin antibodies in HgCl 2 -exposed BXSB mice suggests that mercury is not eliciting a polyclonal B-cell response, nor is it promoting a Th2 response. The acceleration of autoimmunity by mercury is metal-specific: Another immune modulatory metal, nickel, did not accelerate autoimmunity in NZBWF1 or MRL mice (19). Similar experiments comparing the effects of mercury and nickel on BXSB mice also revealed that nickel was ineffective in accelerating autoimmunity in BXSB mice (data not shown). These observations suggest that HgCl 2 is acting as a trigger to complement genetic susceptibility in autoimmune-prone mice rather than simply inducing HgIA.
Maturation of splenic CD4 + T cells from naive to activated phenotype, including increased expression of CD44 and loss of CD62L (44), has been associated with accelerated disease in male BXSB mice (40,44). Increases in such activated CD4 + T cells were also observed in HgCl 2 -exposed female BXSB mice. This appeared to be dosedependent because it was found only in mice given 4 µg HgCl 2 . Male BXSB also develop a strain-specific peripheral blood monocytosis (45). The cells express Mac-1 (CD11b) but not I-A, CD3, or B220 (45), and are thought to contribute to the pathogenesis of lupus in these mice (46). In contrast, increased percentages of Mac-1 + cells are not found in young female BXSB mice (45,46). HgCl 2 exposure increased the percentage of Mac-1 + cells in female BXSB mice, although the levels were low and reached statistical significance only for the group given 4 µg HgCl 2 . Whether this small increase in the percentage of monocytes in the peripheral blood contributes to autoimmunity is questionable, especially because mice given 0.4 µg HgCl 2 had antichromatin antibodies in the peripheral circulation in the absence of any significant changes in PBMCs.
The relevance of these observations to human lupus will require further investigation because considerable debate exists regarding the sources and levels of mercury exposure within the human population (16). Consequently, few studies have attempted to equate environmentally relevant mercury exposure with systemic autoimmune disease  alone. Kidney sections were obtained from mice exposed to HgCl 2 for a total of 38 weeks, starting at 8 weeks of age. Glomeruli in (C) show substantial mesangial matrix expansion, thickening of glomerular capillary tufts, and slight increase in cellularity. Glomeruli in (B) show only minimal changes in the mesangial region. Magnification x400.  (47). Most studies have used levels of mercury to elicit autoimmunity in healthy (8) and autoimmune-prone mice (19) that, although possibly relevant to human occupational exposure (16), are greater than environmental exposure. In attempting to determine the most relevant dose of mercury that would be comparable to environmental exposure, we focused on mercury levels in the kidney. There are several reasons for this: The kidney is a major site of accumulation of mercury in humans and mice (16,48). Reliable data exist on human kidney mercury levels with differing environmental exposures (33,34). Steady-state levels for mercury in mice are achieved after less than 4 weeks' exposure (49). In addition, the kidney is a major target organ in human and murine lupus and may constitute an initial site of tissue damage that leads to systemic exacerbation of disease (50). Levels of mercury in the kidneys in nonoccupationally exposed humans cover a broad range, from undetectable to 2,100 ng Hg/g wet weight of tissue (33,34), with the highest levels being found in dental amalgam bearers [average 433 ng Hg/g wet weight (34)]. The mercury levels in the kidneys of mice exposed to 0.4 µg HgCl 2 /injection fall within the range found in nonoccupationally exposed humans. These mice had accelerated antichromatin antibodies and proteinuria, which suggests that environmentally relevant tissue levels of mercury could be associated with exacerbations of autoimmunity in genetically susceptible hosts.