CD19 expression in B cells is important for suppression of contact hypersensitivity

Abstract

Contact hypersensitivity (CHS) is a cutaneous immune reaction mediated mainly by antigen-specific effector T cells and is regarded as a model for Th1/Tc1-mediated inflammation. However, recent reports have suggested pivotal roles of B cells in CHS. CD19 serves as a positive B-cell response regulator that defines signaling thresholds critical for B-cell responses. In the current study, we assessed the role of the B-cell-specific surface molecule CD19 on the development of CHS by examining CD19-deficient mice. Although CD19-deficient mice are hyposensitive to a variety of transmembrane signals, CD19 loss resulted in increased and prolonged reaction of CHS, suggesting an inhibitory role of CD19 expression in CHS. Sensitized lymph nodes and elicited ear lesions from CD19-deficient mice exhibited Th1/Tc1-shifted cytokine profile with increased interferon-gamma expression and decreased interleukin-10 expression. Adoptive transfer experiments revealed that CD19 expression in recipient mice was required for optimal suppression of CHS response, indicating its role in the elicitation phase. Furthermore, spleen B cells, especially marginal zone B cells, from wild-type mice were able to normalize exaggerated CHS reactions in CD19-deficient mice. Thus, CD19 expression in B cells is critical for termination of CHS responses, possibly through the function of regulatory B cells.

Figure 1

Augmented CHS reaction in CD19^−/− mice. A: Wild-type and CD19^−/− mice were sensitized by epicutaneous application of 0.5% DNFB, and thickness of ear lobes challenged with 0.25% DNFB or carrier (control) 5 days later was measured at indicated time points. B: Wild-type and CD19^−/− mice were sensitized and challenged by application of 0.5% FITC or carrier (control), and thickness of ear lobes were measured. Each experiment included five to seven mice and was repeated at least three times. *P < 0.05, **P < 0.01.

Figure 2

Histopathology of CHS-elicited ear pinnae in wild-type and CD19^−/− mice. Wild-type and CD19^−/− mice were sensitized and challenged by DNFB as in Figure 1A. Representative sections before sensitization with DNFB (A), 24 hours after elicitation (B), and 120 hours after elicitation (C) of ear lobes from wild-type mice (top) and CD19^−/− mice (bottom). The sections were stained with H&E. Original magnifications, ×200.

Figure 3

Profile of infiltrating cells in CHS lesion in wild-type and CD19^−/− mice. The numbers of total infiltrating cells (A), CD4⁺ T cells and CD8⁺ T cells (B), macrophages (D), and polymorphonuclear cells (E) per one field of view (×200) in the DNFB-challenged ear lobes were counted. In C the rate of CD4⁺ T cells and CD8⁺ T cells is shown based on the numbers in B. *P < 0.05, **P < 0.01.

Figure 4

T lymphocytes present in LNs from CD19^−/− mice before or after sensitization. Inguinal LNs were harvested before or after sensitization with DNFB in wild-type and CD19^−/− mice. In A, cells were stained with anti-CD4 and anti-CD8 Abs and analyzed by two-color flow cytometric analysis. Numeric characters (%) indicate the rates of CD4⁺ cells and CD8⁺ cells in total lymphoid cells. In B, Thy1.2⁺ cells were gated and examined for CD4 and CD25 expression by three-color flow cytometric analysis. Numeric characters (%) in B indicate the rate of CD25⁺ cells in CD4⁺ cells in wild-type mice and CD19^−/− mice before and after sensitization. Results represent those obtained with five mice.

Figure 5

Cytokine expression in LNs and ear lobes of wild-type and CD19^−/− mice. Inguinal LNs 2 days after sensitization (A) and ear lobes 24 hours after elicitation (B) were collected from wild-type and CD19^−/− mice (five per group), and RNA was extracted from the specimens. The mRNA levels of IFN-γ, IL-2, IL-12 p40, IL-4, and IL-10 were analyzed by quantitative RT-PCR and normalized with internal control GAPDH. Data are shown as mean ± SEM from five mice. *P < 0.05, **P < 0.01.

Figure 6

Adoptive cell transfer of LN cells in CHS reaction. Whole cells (A), T cells (B), and B cells (C) were prepared from inguinal LNs of sensitized wild-type or CD19^−/− mice and transferred to unsensitized mice as indicated. CHS was elicited 24 hours after transfer using DNFB. Ear thickness was measured as in Figure 1, and the ear swelling values indicate the difference between the ear thickness before and after each time point. Each experiment included three mice and was repeated at least three times. *P < 0.05, **P < 0.01.

Figure 7

In vitro T-cell and DC function in CD19^−/− mice. A: Proliferation of splenic CD4⁺ T cells from CD19^−/− and wild-type mice in response to anti-TCR Ab. CFSE fluorescence intensity of CD4⁺ T cells 5 days after incubation. The rate of cells with reduced fluorescence intensity, ie, T cells with cell division, were regarded as the index reflecting cell proliferation and used in B and C. B: Proliferative response of splenic CD4⁺ T cells from CD19^−/− and wild-type mice by allogeneic DCs in mixed leukocyte reaction assays. Splenic CD4⁺ T cells of C57BL/6 wild-type or CD19^−/− mice were stained with CFSE, mixed with splenic DCs of BALB/c mice, and incubated for 5 days. The rate of the cells with reduced CFSE intensity was measured. C: Capacity of splenic DCs from CD19^−/− and wild-type mice to stimulate allogeneic CD4⁺ T cells in mixed leukocyte reaction assays. Splenic CD4⁺ T cells of BALB/c wild-type mice, stained with CFSE, were mixed with splenic DCs of C57BL/6 wild-type or CD19^−/− mice and incubated for 5 days. The rate of the T cells with reduced CFSE intensity was measured as in B.

Figure 8

Adoptive cell transfer of spleen cells in CHS reaction. Whole splenocytes (A), splenic T cells (B), and splenic B cells (C) were isolated from spleen of sensitized wild-type or CD19^−/− mice, and transferred to unsensitized mice as indicated. CHS was elicited 24 hours after transfer. Ear swelling was assessed as described in Figure 6. Each experiment included three mice and was repeated at least three times. *P < 0.05, **P < 0.01.

Figure 9

Adoptive transfer of sera and B-cell subsets in CHS reaction. Sera, peritoneal B-1 cells, splenic B-2 cells, and splenic MZ B cells from wild-type mice 5 days after sensitization (A) or before sensitization (B) were transferred to CD19^−/− mice. CHS was elicited 24 hours after transfer. Ear swelling was assessed as described in Figure 6. Each experiment included three mice and was repeated at least three times. *P < 0.05, **P < 0.01.

Figure 10

Adoptive transfer of splenic CD19⁺ DCs in CHS reaction. Splenic CD19⁺ DCs from wild-type mice 5 days after sensitization were sorted and then transferred to CD19^−/− mice that were also sensitized 5 days before. PBS-injected wild-type or CD19^−/− mice served as controls. CHS was elicited 24 hours after transfer. Ear swelling was assessed as described in Figure 6. Each experiment included three mice and was repeated at least three times. *P < 0.05, **P < 0.01.