Figure 1.
Increased IgA production in CD37-deficient mice.
(A) Murine CD37 mRNA expression (arbitrary units) on various MACS-purified leukocyte subsets determined by quantitative RT–PCR. i/mDC = (im)mature dendritic cell, mf = macrophage. (B) Human CD37 expression on peripheral blood CD19+ B cells determined by flow cytometry (left) and in B cell follicle area (arrow) of spleen determined by immunohistochemistry (right). Scale bar is 50 µm. Similar CD37 expression was found in lymph nodes. (C) Sera of non-immunized WT (black) and CD37−/− (white) mice were analyzed for the amount of immunoglobulin isotypes by ELISA. Antibody titer is expressed in arbitrary units and represented as mean±SEM (n = 9). Asterisks indicate significant difference (*p<0.0002, **p<0.00005). (D) WT and CD37−/− mice were immunized with NP-KLH and sera were assayed for high affinity NP-specific IgA by ELISA (left). IgA response in chimeric mice that contain either WT or CD37-deficient B cell compartment 35 d after immunization (right). Antibody titer is expressed in arbitrary units and represented as mean±SEM (n = 6). Asterisks indicate significant difference as per: *p<0.03 and **p<0.001. Similar results were obtained for total (low and high affinity) NP-specific IgA.
Figure 2.
Increased formation of IgA–secreting plasma cells in CD37−/− mice.
(A) The frequency of high affinity NP-specific IgA–ASC was assessed in CD37−/− (white) and WT (black) spleen and bone marrow by ELISPOT at d 14 and 35 of the immune response. Non-immunized mice are shown as control (grey). Data are presented as mean±SEM (n = 6). Asterisks indicate significant differences at p<0.05. (B) Immunohistochemical analysis of WT (left) and CD37−/− spleens (right) demonstrating IgA–positive plasma cells (arrows) at d 14 after immunization. Scale bar is 50 µm. (C) Immunohistochemical analysis of serial sections from spleens of CD37−/− mice stained with antibodies specific for IgA (left) and plasma cell marker CD138 (right) at d 14 after immunization. Arrows indicate co-localization. Scale bar is 50 µm (upper) and 15 µm (lower). Mesenteric lymph nodes (D) and Peyer's patches (E) of WT and CD37−/− mice were analyzed by immunohistochemistry at 14 d after NP-KLH immunization using antibodies specific for B cells (upper) and IgA (lower). Note the increased numbers of IgA–positive cells in CD37−/− tissue. Scale bars are 300 µm. (F) Percentage of IgA+ and IgG1+ NP-specific plasma cells in Peyer's patches of WT (black) and CD37−/− (white) mice determined by flow cytometry at d 21 after immunization. Data are presented as mean±SEM (n = 4). Asterisk indicates statistical difference at p<0.05.
Figure 3.
Increased IgA production after immunization in CD37−/− mice is dependent on IL-6.
(A) Immunohistochemical analysis of spleens from CD37−/− and WT mice 14 d after NP-KLH immunization. Germinal centers (identified in serial sections by PNA staining) are indicated in the B cell follicles. Immunized WT mice have non-detectable levels of IL-6 in spleens (left). In contrast, B cell follicles in spleens of immunized CD37−/− mice are clearly positive for IL-6 (red) in the GC area (right). Spleens of non-immunized mice (WT and CD37−/−) were negative for IL-6 staining (not shown). Scale bar is 50 µm. (B) Effect of neutralizing anti–IL-6 during ex vivo restimulation. Splenocytes from WT and CD37−/− mice were prepared 14 d after NP-KLH immunization, and stimulated in vitro with NP-KLH (1 µg/ml) in the absence or presence of anti–IL-6. Supernatants were collected after 48 h, and assayed for IgA production by ELISA (expressed in arbitrary units). Asterisk indicates significant difference (*p<0.002). (C) IL-6 was neutralized in WT and CD37−/− mice during immunizations using blocking IL-6 antibodies (as described in Materials and methods). High affinity NP-specific IgA was assayed in serum of CD37−/− mice treated with anti–IL-6 (black) or control antibody (white) (left). Antibody titer is expressed in arbitrary units and represented as mean±SEM (n = 6). Asterisks indicate significant difference as per: *p<0.04. Histogram shows percentage of CD37−/− mice with high IgA anti-NP3 levels (above 10× background level) in serum after treatment with anti–IL-6 (black) compared to control treated CD37−/− mice (white) at indicated days after immunization (right). Similar results were obtained for total NP-specific antibody (against NP20-BSA).
Figure 4.
CD37−/− mice are protected against C. albicans infection.
CD37−/− and WT mice (n = 5) were systemically infected with 1×105 C. albicans. (A) Spleens were removed 3 or 7 d after infection and splenocytes were stimulated with live, heat-killed C. albicans yeasts, zymosan (E∶T ratio 2∶1) for 48 h, after which IL-6, IL-10, IL-17, and γIFN in supernatants were measured by ELISA. Asterisks indicate significant difference as per: *p<0.05 and **p<0.002. (B) Serum of CD37−/− and WT mice was analyzed for the presence of IgA antibodies reactive with C. albicans yeast (left) or zymosan (right) by flow cytometry as described in Materials and methods. Serum of non-infected mice is shown as control. Asterisks indicate significant difference as per: *p<0.05. (C) left: Subgroups of five or six animals were sacrificed on d 1 or 7, and kidneys were analyzed for the number of viable Candida cells (expressed as log CFUg−1 tissue). Horizontal bars represent mean. Asterisks indicate significant difference as per: *p<0.0005. Right: Histology of kidneys from WT and CD37−/− mice 7 d after infection. Note the major infection area and infiltrating hyphae in WT kidneys. (D) Survival of WT and CD37−/− mice (n = 6) was assessed after intravenous injection of lethal dose of 5×105 CFU C. albicans.
Figure 5.
Adoptive transfer of CD37−/− serum increases resistance of WT mice to fungal infection.
(A) Percentage of GR-1+ blood leukocytes that phagocytosed FITC-labeled C. albicans in the absence or presence of serum from infected WT or CD37−/− mice for various time points at 37°C was quantified by flow cytometry. Sera were heat-inactivated (56°C for 30 min) to inactivate complement (left). In addition, uptake in the presence of WT or CD37−/− serum was analyzed by fluorescence light microscopy (right; data shown for 30-min. incubation). Bar represents 10 µm. (B) C. albicans was incubated in the absence or presence of 20% serum (from infected WT or CD37−/− mice) or 20% IgA–depleted serum and growth (CFU) was assessed. Asterisks indicate significant difference as per: p<0.005. (C) WT mice (n = 5) were systemically infected with 1×105 C. albicans and untreated, or treated with either pooled CD37−/− serum containing high levels of fungal-specific IgA, or IgA-depleted CD37−/− serum. Kidneys were analyzed for the number of viable Candida cells (expressed as log CFUg−1 tissue) 7 d after infection. Horizontal bars represents mean. Asterisks indicate significant difference as per: p<0.05.