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Increased B cell deletion and significantly reduced auto-antibody titre due to premature expression of human complement receptor 2 (CR2, CD21).

Pappworth IY, Kulik L, Haluszczak C, Reuter JW, Holers VM, Marchbank KJ - Mol. Immunol. (2009)

Bottom Line: We found that expression of hCR2 on the B6(lpr) background resulted in a significant reduction in levels of anti-nuclear antibodies (ANA) generated as mice aged but the levels of ANA were still higher than those found in age matched C57BL/6j (B6) mice.B cells from hCR2(high) mice were found to display a higher baseline level of apoptosis, whether analysed ex vivo or after in vitro culture, than their B6 counterparts and this was apparently linked to both surface IgM expression by the B cells and C3 levels in the mice.Overall, we have demonstrated that mice expressing hCR2 on their B cells during bone marrow development display a higher degree of apoptosis which may lead to a deletion of autoreactive B cells and be protective against the development of autoimmune disease.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Newcastle University, Central Parkway, Center for Life, Newcastle-upon-Tyne NE1 3BZ, UK.

ABSTRACT
The involvement of complement receptor 2 (CR2) in B cell tolerance and autoimmune disease has been revealed over the past decade or so. Our previous studies have established that mice prematurely expressing human CR2 under the control of a lambda light chain promoter (in particular the hCR2(high) line) have a marked deficit in their immune response to various antigens and fail to develop collagen-induced arthritis. This phenotype appears to be the result of irreversible changes in B cell signalling pathways and suggested that hCR2 expressing mice are protected from developing autoimmune disease. To test this hypothesis, we examined the ability of the hCR2 to block the development of spontaneous autoimmune disease on the C57BL/6j-Fas(lpr/)Fas(lpr) (B6(lpr)) background. We found that expression of hCR2 on the B6(lpr) background resulted in a significant reduction in levels of anti-nuclear antibodies (ANA) generated as mice aged but the levels of ANA were still higher than those found in age matched C57BL/6j (B6) mice. B cells from hCR2(high) mice were found to display a higher baseline level of apoptosis, whether analysed ex vivo or after in vitro culture, than their B6 counterparts and this was apparently linked to both surface IgM expression by the B cells and C3 levels in the mice. Our data also provides evidence that B cell survival in the presence of hCR2 is heavily modified by the background strain of the mouse. Overall, we have demonstrated that mice expressing hCR2 on their B cells during bone marrow development display a higher degree of apoptosis which may lead to a deletion of autoreactive B cells and be protective against the development of autoimmune disease.

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B cells from hCR2high mice express less hCR2 after sIgM expression in the presence of C3. BM cells were collected from C3+/+hCR2high and C3−/−hCR2high mice as described in materials and methods. BM lymphocytes were stained for B220+ and hCR2 followed by staining for IgM before (Extracellular) or after (Intracellular) incubation with fix/perm (BD biosciences, Oxford, UK). Dot plots show B220-FITC+ cells expressed as hCR2-PE (Bly-4) versus anti-IgM-Cy5.5. Results shown are representative of 3 independent experiments involving 10 mice from each genotype in total.
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fig3: B cells from hCR2high mice express less hCR2 after sIgM expression in the presence of C3. BM cells were collected from C3+/+hCR2high and C3−/−hCR2high mice as described in materials and methods. BM lymphocytes were stained for B220+ and hCR2 followed by staining for IgM before (Extracellular) or after (Intracellular) incubation with fix/perm (BD biosciences, Oxford, UK). Dot plots show B220-FITC+ cells expressed as hCR2-PE (Bly-4) versus anti-IgM-Cy5.5. Results shown are representative of 3 independent experiments involving 10 mice from each genotype in total.

Mentions: We have previously shown that hCR2 expression levels drop significantly as the B cells mature from B220lo to B220hi and are reduced further still as B cells migrate into the periphery (Birrell et al., 2005). On analysis of B cells isolated from C3−/− hCR2high mice, we found that this reduction in hCR2 expression levels was no longer apparent, essentially leading to an apparent 3-fold increase in hCR2 expression levels on mature B cells in conjunction with an improvement in the number of B cells reaching the periphery (Twohig et al., 2007). These previous data, together with the data showing that apoptosis levels in the BM of C3−/−hCR2high were reduced compared with hCR2high mice (Fig. 2c), led us to look more closely at the factors involved in the down regulation or selection of cells with low levels of hCR2 in the presence of C3. Examination of BM B cells for intracellular and extracellular expression of sIgM, in conjunction with hCR2 surface expression, demonstrated that sIgM expressing cells had lower levels of hCR2 on the cell surface than B cells which had constructed but not expressed sIgM (Fig. 3). These results were confirmed using several different antibodies to hCR2 in order to control for the potential loss or blocking of antibody binding sites due to the presence and binding of newly formed C3 fragments to CR2 in the C3 sufficient mice (data not shown). When this analysis was subsequently carried out in the C3−/− background, we found that hCR2 expression levels on cells that expressed sIgM were not significantly different from those before sIgM was expressed. Overall, these data suggest that the levels of hCR2 are tightly regulated on the mouse B cell at the point of sIgM expression and that C3 and/or its break down components play an important part in this mechanism.


Increased B cell deletion and significantly reduced auto-antibody titre due to premature expression of human complement receptor 2 (CR2, CD21).

Pappworth IY, Kulik L, Haluszczak C, Reuter JW, Holers VM, Marchbank KJ - Mol. Immunol. (2009)

B cells from hCR2high mice express less hCR2 after sIgM expression in the presence of C3. BM cells were collected from C3+/+hCR2high and C3−/−hCR2high mice as described in materials and methods. BM lymphocytes were stained for B220+ and hCR2 followed by staining for IgM before (Extracellular) or after (Intracellular) incubation with fix/perm (BD biosciences, Oxford, UK). Dot plots show B220-FITC+ cells expressed as hCR2-PE (Bly-4) versus anti-IgM-Cy5.5. Results shown are representative of 3 independent experiments involving 10 mice from each genotype in total.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657831&req=5

fig3: B cells from hCR2high mice express less hCR2 after sIgM expression in the presence of C3. BM cells were collected from C3+/+hCR2high and C3−/−hCR2high mice as described in materials and methods. BM lymphocytes were stained for B220+ and hCR2 followed by staining for IgM before (Extracellular) or after (Intracellular) incubation with fix/perm (BD biosciences, Oxford, UK). Dot plots show B220-FITC+ cells expressed as hCR2-PE (Bly-4) versus anti-IgM-Cy5.5. Results shown are representative of 3 independent experiments involving 10 mice from each genotype in total.
Mentions: We have previously shown that hCR2 expression levels drop significantly as the B cells mature from B220lo to B220hi and are reduced further still as B cells migrate into the periphery (Birrell et al., 2005). On analysis of B cells isolated from C3−/− hCR2high mice, we found that this reduction in hCR2 expression levels was no longer apparent, essentially leading to an apparent 3-fold increase in hCR2 expression levels on mature B cells in conjunction with an improvement in the number of B cells reaching the periphery (Twohig et al., 2007). These previous data, together with the data showing that apoptosis levels in the BM of C3−/−hCR2high were reduced compared with hCR2high mice (Fig. 2c), led us to look more closely at the factors involved in the down regulation or selection of cells with low levels of hCR2 in the presence of C3. Examination of BM B cells for intracellular and extracellular expression of sIgM, in conjunction with hCR2 surface expression, demonstrated that sIgM expressing cells had lower levels of hCR2 on the cell surface than B cells which had constructed but not expressed sIgM (Fig. 3). These results were confirmed using several different antibodies to hCR2 in order to control for the potential loss or blocking of antibody binding sites due to the presence and binding of newly formed C3 fragments to CR2 in the C3 sufficient mice (data not shown). When this analysis was subsequently carried out in the C3−/− background, we found that hCR2 expression levels on cells that expressed sIgM were not significantly different from those before sIgM was expressed. Overall, these data suggest that the levels of hCR2 are tightly regulated on the mouse B cell at the point of sIgM expression and that C3 and/or its break down components play an important part in this mechanism.

Bottom Line: We found that expression of hCR2 on the B6(lpr) background resulted in a significant reduction in levels of anti-nuclear antibodies (ANA) generated as mice aged but the levels of ANA were still higher than those found in age matched C57BL/6j (B6) mice.B cells from hCR2(high) mice were found to display a higher baseline level of apoptosis, whether analysed ex vivo or after in vitro culture, than their B6 counterparts and this was apparently linked to both surface IgM expression by the B cells and C3 levels in the mice.Overall, we have demonstrated that mice expressing hCR2 on their B cells during bone marrow development display a higher degree of apoptosis which may lead to a deletion of autoreactive B cells and be protective against the development of autoimmune disease.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Newcastle University, Central Parkway, Center for Life, Newcastle-upon-Tyne NE1 3BZ, UK.

ABSTRACT
The involvement of complement receptor 2 (CR2) in B cell tolerance and autoimmune disease has been revealed over the past decade or so. Our previous studies have established that mice prematurely expressing human CR2 under the control of a lambda light chain promoter (in particular the hCR2(high) line) have a marked deficit in their immune response to various antigens and fail to develop collagen-induced arthritis. This phenotype appears to be the result of irreversible changes in B cell signalling pathways and suggested that hCR2 expressing mice are protected from developing autoimmune disease. To test this hypothesis, we examined the ability of the hCR2 to block the development of spontaneous autoimmune disease on the C57BL/6j-Fas(lpr/)Fas(lpr) (B6(lpr)) background. We found that expression of hCR2 on the B6(lpr) background resulted in a significant reduction in levels of anti-nuclear antibodies (ANA) generated as mice aged but the levels of ANA were still higher than those found in age matched C57BL/6j (B6) mice. B cells from hCR2(high) mice were found to display a higher baseline level of apoptosis, whether analysed ex vivo or after in vitro culture, than their B6 counterparts and this was apparently linked to both surface IgM expression by the B cells and C3 levels in the mice. Our data also provides evidence that B cell survival in the presence of hCR2 is heavily modified by the background strain of the mouse. Overall, we have demonstrated that mice expressing hCR2 on their B cells during bone marrow development display a higher degree of apoptosis which may lead to a deletion of autoreactive B cells and be protective against the development of autoimmune disease.

Show MeSH
Related in: MedlinePlus