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. 1996 Dec 1;184(6):2271-8.
doi: 10.1084/jem.184.6.2271.

Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice

S D Wolf  1 , B N Dittel, F Hardardottir, C A Janeway Jr
Affiliations

Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice

S D Wolf et al. J Exp Med. .

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an animal model for autoimmune central nervous system disease mediated by CD4 T cells. To examine the role of B cells in the induction of EAE, we used B10.PL (I-Au) mice rendered deficient in B cells by deletion of their mu chain transmembrane region (B10.PLmicroMT). By immunizing B10.PL and B10.PLmicroMT mice with the NH-terminal myelin basic protein encephalitogenic peptide Ac1-11, we observed no difference in the onset or severity of disease in the absence of mature B cells. There was, however, a greater variation in disease onset, severity, and especially of recovery in the B cell-deficient mice compared to controls. B10.PLmicroMT mice rarely returned to normal in the absence of B cells. Taken together, our data suggest that B cells do not play a role in the activation of encephalitogenic T cells, but may contribute to the immune modulation of acute EAE. The mechanisms to explain these effects are discussed.

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Figures

Figure 1
Figure 1
Two-color dot plots of Ig and TCR αβ expression on spleen cells from B10.PL and B10.PLμMT mice. Spleen cells were stained for the expression of Ig (x-axis) and αβ TCR (y-axis). Data represent analysis performed on male age matched B10.PL (A) and B10.PLμMT (B) mice from one of five sets of experiments each with similar results.
Figure 2
Figure 2
Proliferation of CD4+ T cells from B10.PL and B10.PLμMT mice in response to Con A and anti-CD3. CD4+ spleen cells from one B10.PL mouse (open square) and one B10.PLμMT mouse (closed circle) were cultured in the presence of 1:3 dilutions of plate-bound anti-CD3 from 10 to 0.12 μg/ ml (A) or 1:3 dilutions of CD4+ T cells from 0.1 to 3.0 × 105 cells/well in the presence of 5 μg/ml Con A (B). Proliferation was measured by [3H]TdR incorporation and is presented as CPM performed in duplicate. Data in A and B are from the same mice with identical animals sharing symbols and represents one third of the experiments, each with similar results.
Figure 3
Figure 3
Proliferation of CD4+ LN cells from B10.PL and B10.PLμMT mice immunized with MBP Ac 1-11 (A) or HEL 30-53 (B). CD4+ LN cells from B10.PL (open squares) and B10.PLμMT (closed circles) were isolated as described in the Material and Methods from the popliteal LN of mice immunized in the footpads with 150 μg MBP Ac1-11 or HEL 30-53 10 d before, and cocultured with splenic APC in the presence of 1:5 dilutions of MBP Ac1-11 or HEL 30-53 from 100 to 0.16 μg/ml. Background proliferation in the absence of stimulating peptide was subtracted from each data point and is shown as zero on the proliferation curves and was less than 10,000 cpm. Proliferation was measured by [3H]TdR incorporation and presented as counts per minute performed in duplicate. (C) CD4+ LN cells from the identical mice used in A and B were incubated with splenic APC as described in the Material and Methods in the presence of 100 μg/ml MBP Ac1-11 or HEL 30-53. After 48 h, total cellular RNA was isolated from 3.2 × 106 cells. RT-PCR was then performed using primers for HPRT and the HPRT product was quantitated to ensure equal levels of cDNA used in subsequent PCR reactions. PCR was then performed using cDNA from 2 × 105 cell equivalents for IL-2, IL-4, IFN-γ, and TNF-α for 30 cycles. The HPRT-PCR reaction was performed with 25 cycles. D10, a Th2 clone, was used as a positive control for IL-4, and D10.TCR 25, a Th1 clone, was used as a positive control for IL-2, IFN-γ, and TNF-α.
Figure 3
Figure 3
Proliferation of CD4+ LN cells from B10.PL and B10.PLμMT mice immunized with MBP Ac 1-11 (A) or HEL 30-53 (B). CD4+ LN cells from B10.PL (open squares) and B10.PLμMT (closed circles) were isolated as described in the Material and Methods from the popliteal LN of mice immunized in the footpads with 150 μg MBP Ac1-11 or HEL 30-53 10 d before, and cocultured with splenic APC in the presence of 1:5 dilutions of MBP Ac1-11 or HEL 30-53 from 100 to 0.16 μg/ml. Background proliferation in the absence of stimulating peptide was subtracted from each data point and is shown as zero on the proliferation curves and was less than 10,000 cpm. Proliferation was measured by [3H]TdR incorporation and presented as counts per minute performed in duplicate. (C) CD4+ LN cells from the identical mice used in A and B were incubated with splenic APC as described in the Material and Methods in the presence of 100 μg/ml MBP Ac1-11 or HEL 30-53. After 48 h, total cellular RNA was isolated from 3.2 × 106 cells. RT-PCR was then performed using primers for HPRT and the HPRT product was quantitated to ensure equal levels of cDNA used in subsequent PCR reactions. PCR was then performed using cDNA from 2 × 105 cell equivalents for IL-2, IL-4, IFN-γ, and TNF-α for 30 cycles. The HPRT-PCR reaction was performed with 25 cycles. D10, a Th2 clone, was used as a positive control for IL-4, and D10.TCR 25, a Th1 clone, was used as a positive control for IL-2, IFN-γ, and TNF-α.
Figure 4
Figure 4
Comparison of EAE clinical course in B10.PL versus B10.PLμMT mice. B10.PL and B10.PLμMT mice were immunized with 150 μg MBP Ac1-11 in the flanks and intravenously injected with pertussis toxin on the day of immunization, and again 48 h later. The individual mice were scored for the severity of EAE using the following scale: 0, no disease; 1, limp tail; 2, hind limb paresis; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, death. Mice were examined daily from day 8 to 40, and the daily score from all B10.PL mice (open squares) and B10.PLμMT mice (closed circles) that developed signs of clinical disease were averaged (A). The data in A represents the average of 16/19 B10.PL mice and 19/22 B10.PLμMT mice that manifested disease. Data from individual mice were plotted from three consecutive B10.PL (squares) (B) and four consecutive B10.PLμMT (circles) (C) mice. The data was collected from one fifth of the experiments set up.
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