BAFF Augments Certain Th1-Associated Inflammatory Responses
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免疫学杂志 2005年第9期
Abstract
B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.
Introduction
Bcell-activating factor belonging to the TNF family (BAFF; 4 BLyS, TALL-1, zTNF-4, THANK) is a TNF superfamily member that plays an important role in immune responses (1, 2). Excessive production of BAFF is associated with the development of autoimmune diseases, because transgenic (Tg) mice that overproduce BAFF develop severe autoimmune disorders that resemble systemic lupus erythematosus and Sj?gren’s syndrome (3, 4, 5, 6). Also, high levels of BAFF have been found in the blood of patients with autoimmune diseases, particularly systemic lupus erythematosus and Sj?gren’s syndrome (5, 7, 8, 9). Thus, excess BAFF may cause a breakdown in immune tolerance through inappropriate survival signals to splenic and peripheral B cells (10, 11, 12). In the spleen, BAFF promotes B cell maturation, particularly at the critical T1-T2 immune tolerance checkpoint (10), and splenic B cells in BAFF-deficient mice fail to mature past the T1 stage (13). Autoreactive B cells are expanded in lymphoid tissues of BAFF Tg mice (3, 14), possibly as a result of BAFF rescue of autoreactive cells that would normally be deleted (12, 14). BAFF also enhances the survival of plasmablasts (15, 16) and stimulates peripheral B cell proliferation, Ab production, and class switching (17, 18, 19).
BAFF binds three receptors: BAFF-R, transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA) (1). BAFF-R is the predominant BAFF receptor on circulating B cells (18) and is also important for the regulation of B cell maturation in the spleen, because BAFF-R-deficient mice show disrupted B cell maturation, similar to that seen in BAFF-deficient mice (13, 20). BAFF-induced survival is mediated by up-regulation of the antiapoptotic molecule Bcl-2 (3), and Bcl-2 overexpression restores B cell survival in the absence of BAFF (21). The other receptors, TACI and BCMA, have restricted patterns of expression on specific B cell subsets (18); BCMA appears to be particularly important for survival or stimulation of plasmablasts and germinal center cells (15, 18), whereas TACI most likely serves as a negative regulator of B cell activity, particularly on activated B cells (22).
More recently, BAFF has emerged as a regulator of T cell function. BAFF provides costimulatory signals to T cells in the presence of suboptimal TCR stimulation and enhances T cell proliferation and cytokine production (18, 23, 24). TACI-Ig and BAFF-R-Ig decoy receptors inhibit T cell activation in vitro (18, 23) and in vivo (25), and BAFF-deficient mice show impaired T cell-mediated allograft rejection (26). Thus, physiological levels of BAFF appear necessary for the generation of optimal T cell responses. These effects occur through BAFF-R, because T cells from BAFF-R mutant mice fail to respond to BAFF, and BAFF-R expression (but not that of TACI or BCMA) is present on the surface of subsets of central and effector memory T cells (18). These findings suggest that BAFF may play a role in stimulating T cell function in vivo, particularly during T cell-mediated pathogenic reactions. Interestingly, BAFF is highly up-regulated in astrocytes in multiple sclerosis plaques (27) and is also produced by fibroblast-like synoviocytes from rheumatoid arthritis patients (28). Both of these autoimmune diseases have a strong T cell association.
CD4+ effector T cells often polarize to either Th1 or Th2 phenotypes (29). Th1 responses control intracellular pathogens such as viruses and are associated with the production of IFN-, IL-2, and TNF- and recruitment of phagocytic leukocytes. In contrast, Th2 responses control infections by large extracellular parasites, in part through production of IL-4, IL-5, and IL-13, and recruitment of eosinophils. Dysregulation of Th1 or Th2 responses may contribute to the pathogenesis of autoimmune diseases as well as allergic diseases such as asthma. The mechanisms that initiate and maintain polarization of T cell responses to Th1 or Th2 are incompletely understood, but instructive signals in the form of cytokines and cell surface molecules expressed by APC are important (30, 31, 32, 33). Th1 and Th2 responses counteract each other, in that strong polarization to a Th1 response suppresses Th2 responses and vice versa (34). Given the ability of BAFF to regulate T cell activation and its association with autoimmune diseases, we investigated whether BAFF affects the outcome of Th1- and Th2-mediated T cell responses in vivo.
We show that systemic overexpression of BAFF in BAFF Tg mice exacerbates the severity of Th1-mediated delayed-type hypersensitivity (DTH) responses by enhanced T cell proliferation and IFN- production in local lymph nodes. Enhanced DTH responses in BAFF Tg mice correlated directly with levels of BAFF in serum, suggesting that the varying levels of BAFF seen in the blood of human subjects might also relate to varying capacities for DTH Th1 responses. High levels of BAFF in BAFF Tg mice also resulted in subdued Th2-mediated responses in an allergic airways disease model. The effects of BAFF on Th1/Th2 responses operated at multiple levels, because enhanced Th1 responses depended on B cells, whereas BAFF inhibition of Th2 responses was B cell independent. These findings demonstrate that BAFF augments certain Th1 responses in vivo, but not Th2 responses, and illustrate the importance of BAFF for T cell as well as B cell responses.
Materials and Methods
Reagents and Abs
Methylated BSA (mBSA) and OVA were purchased from Sigma-Aldrich. Imject CFA and Imject alum were purchased from Pierce. All anti-mouse FITC-, PE-, CyChrome-, PerCP-, and allophycocyanin-conjugated mAbs were obtained from BD Pharmingen. Murine IL-4, IL-5, IFN-, and IL-10 ELISA kits were purchased from BD Biosciences. Goat anti-mouse alkaline phosphatase-conjugated Abs were purchased from Southern Biotechnology Associates. Human BCMA-Ig was provided by Biogen-Idec and has been described previously (35).
Mice
Animals were housed under conventional barrier protection and handled in accordance with the animal experimentation ethics committee, which complies with the Australian code of practice for the care and use of animals for scientific purposes. BAFF Tg mice (3) and BAFF–/– mice (13) were supplied by Biogen-Idec and were bred in our animal facility. BAFF Tg mice were maintained as mice homozygous for the BAFF transgene, and wild-type (WT) mice were used as controls, as previously described (36). Two lines of BAFF Tg mice were used for experimentation, referred to as lines 1 and 2, which differ slightly in BAFF levels and immune responses (see below). μMT–/– mice (37) were purchased from The Jackson Laboratory. μMT–/– x BAFF Tg mice were established by breeding homozygous μMT–/– and BAFF Tg line 1 mice and subsequent breeding of the F1 generation. Homozygous μMT–/– x BAFF Tg mice and WT controls were derived from the F2 generation and bred as separate lines for experimentation. All mice were maintained on a C57BL/6 background
DTH responses
Six- to 8-wk-old mice were injected s.c. at the tail base with 200 μl of 1.25 mg/ml mBSA in CFA (1/1 mix) on day 1, as described previously (38). On day 7, mice were rechallenged in one hind footpad with an injection of 20 μl of a 10 mg/ml mBSA solution, whereas the opposite hind footpad received 20 μl of 1x PBS. Paw swelling was measured 8–72 h after rechallenge with a dial thickness gauge. For paw tissue section analysis, mice were killed at 48 h after challenge. The paws of mice were dissected and fixed in 10% formalin (Sigma-Aldrich) in PBS for 5 days. Paws were decalcified for 10% formalin in PBS supplemented with formic acid (Sigma-Aldrich) at a 9:1 ratio before being embedded in paraffin, sectioned, and stained with H&E.
For analysis of mBSA-specific Abs, mice were killed 7 days after footpad challenge, serum was collected, and mBSA-specific Abs levels were measured by ELISA. In vitro recall responses were measured by collecting inguinal lymph nodes 7 days after immunization. Cultures were normalized to 2 x 106 T cells/ml and were restimulated for 72 h with 40 μg/ml mBSA in X-Vivo 15 serum-free medium (BioWhittaker) supplemented with penicillin/streptomycin. Supernatants were collected for measurement of cytokine production by ELISA. Proliferation was measured by addition of 1 μCi of [3H]thymidine (Amersham Biosciences)/well 18 h before harvesting and subsequent beta scintillation counting.
OVA-induced allergic airway inflammation
Six- to 8-wk-old mice were injected i.p. with 200 μl of a 1 mg/ml OVA solution with alum (1/1 mix) on days 1 and 15. Mice were given a 20-min aerosol on days 28, 30, 32, and 34 consisting of either PBS or 1% OVA in PBS. Mice were killed on day 35, and bronchoalveolar lavage (BAL) fluid and organs were collected. Total BAL fluid cell numbers were enumerated, and differential cell counts were performed after cytospin and Giemsa staining to establish the number and identity of infiltrating cells. Lung tissues were fixed in 10% Formalin/PBS for 7 days and embedded in paraffin. Standard protocols for H&E staining were used to stain tissue sections. At the completion of the aerosol regimen, peribronchial lymph nodes were collected, pooled into groups, and cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS and penicillin/streptomycin. Cultures were normalized to 2 x 106 T cells/ml and restimulated for 72 h with 100 μg/ml OVA, and proliferation and cytokine production were measured as per DTH. In vitro recall responses were measured by immunizing mice at the tail base with 200 μl of 1 mg/ml OVA in alum (1/1 mix) and collecting inguinal lymph nodes 7 days later. Cells were restimulated in vitro as described above for peribronchial lymph nodes, using 500 μg/ml OVA.
ELISA
The mouse BAFF ELISA was performed as previously described (36). The Ab response to mBSA was analyzed as follows. MaxiSorb plates (Nalge Nunc International; 384-well) were coated with 50 μg/ml mBSA diluted in 0.1 M sodium bicarbonate buffer (pH 9.6) at 4°C overnight. Plates were washed three times with PBS-0.05% Tween 20. Serial dilutions of mouse serum in ELISA buffer (1% BSA in PBS) were added to the plate. Anti-mBSA Abs were detected using 0.125 μg/ml alkaline phosphatase-conjugated goat anti-mouse IgM, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, or anti-IgA (Southern Biotechnology Associates). p-Nitrophenyl phosphate tablets (Sigma-Aldrich) were used for detection, and plates were read at an OD of 405 nm. The titer (log base 2) is defined as the serum dilution giving an OD four times higher than that of background (where 1 = 1/50 dilution).
Statistical analysis
Statistical significance was determined using t test, and significance with relation to comparison data is indicated by p values in the figure legends.
Results
Enhanced DTH responses in BAFF Tg mice
Six- to 8-wk-old mice were immunized intradermally at the base of the tail with mBSA in CFA and challenged 7 days later with an s.c. injection of mBSA into the footpad. BAFF Tg mice from two distinct transgenic lines (lines 1 and 2) displayed significantly increased paw swelling compared with WT mice (Fig. 1, A and B). Paw swelling was comparable in WT and BAFF Tg mice at early time points after challenge (Fig. 1A), but was significantly higher in BAFF Tg mice 48 and 72 h after challenge. DTH responses in BAFF Tg mice at 48 h were also characterized by increased erythema (Fig. 1B), and histological analysis of paw tissue sections revealed an increased cellular infiltrate into the footpads (Fig. 1C). Serum anti-mBSA Ab isotypes measured 7 days after footpad challenge showed BAFF Tg mice to have significantly higher titers of mBSA-specific IgG1, IgG2a, and IgG2b compared with WT mice (Fig. 1D), whereas mBSA-specific IgA levels showed a nonsignificant trend toward an increase (data not shown). Levels of mBSA-specific IgM and IgG3 were similar in WT and BAFF Tg mice (data not shown).
FIGURE 1. BAFF Tg mice show increased DTH responses. A, Increased paw swelling over time in two lines of BAFF Tg mice (n = 10, line 1; n = 6, line 2). B, Increased erythema in paws of BAFF Tg mice at 48 h compared with WT mice. C, H&E staining of paw sections at 48 h reveals increased leukocyte infiltration in BAFF Tg mice compared with WT controls. D, Increased serum levels of mBSA-specific Abs in BAFF Tg mice 7 days after footpad challenge (n = 11, WT; n = 10, BAFF Tg). *, p < 0.05; **, p < 0.01; ***, p < 0.005.
BAFF levels determine the magnitude of the DTH response
The concentration of BAFF in human blood varies considerably; most individuals express low levels, but some autoimmune patients express levels >100 ng/ml (5). BAFF Tg mice also show variable levels of BAFF in blood due to secondary endogenous BAFF production in response to autoimmune activation (36). To assess the relationship between serum BAFF levels and paw swelling, serum from both WT and BAFF Tg mice was collected 48 h after challenge, and the level of BAFF in serum was determined by ELISA. Both lines of BAFF Tg mice displayed high levels of BAFF in serum (line 1 average concentration, 215.03 ± 50.69 ng/ml; line 2 average concentration, 1128.2 ± 315.8 ng/ml), whereas lower levels of BAFF were detected in WT mice (5.27 ± 1.47 ng/ml), similar to levels in unimmunized mice (36). When the level of BAFF in serum was correlated with paw swelling for each mouse (Fig. 2A), a strong correlation was observed (r2 = 0.593). Thus systemic BAFF levels may regulate the magnitude of certain T cell responses.
FIGURE 2. DTH responses correlate with serum BAFF levels, although BAFF deficiency does not eliminate DTH responses. A, Serum levels of BAFF in BAFF Tg mice were measured using ELISA and plotted against paw swelling 48 h after footpad rechallenge. B, DTH responses in BAFF–/– mice, and mice heterozygous for the knockout allele (n = 6). C, C57/B6 mice treated with BCMA-Ig showed no reduction in DTH severity compared with control (PBS-treated) mice (n = 6).
We next examined DTH responses in BAFF-deficient mice. These mice were competent in mounting a normal DTH response (Fig. 2B). Also, WT mice treated with BCMA-Ig showed no consistent reduction in DTH responses compared with untreated mice (Fig. 2C). Thus, BAFF is not essential for basal DTH responses, but in circumstances of BAFF overproduction, the magnitude of DTH responses correlates with the level of BAFF.
Increased mBSA-specific T cell proliferation and IFN- production in BAFF Tg mice
The mechanisms underlying the enhanced DTH response in BAFF Tg mice were investigated, first by examining T cell responses to mBSA in vitro. WT and BAFF Tg mice were immunized at the tail base as described above, and inguinal lymph nodes were collected 7 days later. Lymph node cells were restimulated in vitro with mBSA for 72 h. T cells from BAFF Tg mice gave a significantly stronger recall response compared with WT mice, displaying a 3-fold increase in proliferation (Fig. 3A) and a 10-fold higher IFN- production in culture supernatants (Fig. 3B). IL-4 and IL-5 levels were below the level of detection in both WT and BAFF Tg mice (data not shown). Thus, in an environment of high BAFF levels, T cell responses to Ag are augmented, and this might be due to qualitative changes in the T cells involved or an increased frequency of Ag-specific memory or effector T cells.
FIGURE 3. Increased mBSA-specific in vitro recall responses by T cells in BAFF Tg mice. A, Mice were injected at the tail base with 250 μg of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 40 μg/ml mBSA for 72 h. Proliferation was measured by thymidine uptake. B, IFN- levels from culture supernatants were measured by ELISA (n = 3). *, p < 0.05.
Numbers of effector memory T cells are increased in BAFF Tg mice, but not in μMT–/– x BAFF Tg mice
We also examined changes in the make-up of the T cell immune system that result from long term exposure to high levels of BAFF and effects that expanded subsets of B cells could have on T cell and DTH responses in BAFF Tg mice. Previous studies of BAFF Tg mice revealed altered CD4+ T cell subset ratios (3). Splenocytes from WT and BAFF Tg mice were isolated and stained with Abs to CD4, CD44, and CD62L. We observed a substantial increase in the proportion of CD44highCD62Llow effector memory CD4+ cells and a decrease in CD44lowCD62Lhigh naive CD4+ T cells in BAFF Tg mice (Fig. 4A, top panels). Functionally, effector memory T cells are closely related to effector T cells and have the capacity to migrate to peripheral tissues (39, 40). To investigate whether the expansion of mature B cell subsets seen in BAFF Tg mice (3) might in some way connect to the increased numbers of effector memory CD4+ T cells, we generated B cell-deficient BAFF Tg (μMT–/– x BAFF Tg) mice, and these showed no increase in effector memory T cell numbers (Fig. 4A, bottom panels). The ratio of effector/naive T cells in line 2 BAFF Tg mice was increased 4-fold (Fig. 4B, left panel), and line 1 BAFF Tg mice also showed a statistically significant (although smaller) increase (Fig. 4B, right panel). Analysis of μMT–/– mice showed a reduced ratio, indicating an overall reduction in effector cells, which was not increased in μMT–/– x BAFF Tg mice (line 1). Thus, the altered makeup of the T cell compartment in BAFF Tg mice is dependent on B cells.
FIGURE 4. Altered T cell phenotype and increased DTH severity in BAFF Tg mice are B cell dependent. A, Splenocytes were stained with anti-CD4, anti-CD44, and anti-CD62L (CD4+ gated cells are shown; n = 3 for all genotypes with a representative plot for each genotype shown). B, Cell ratios for CD4+ T cells calculated based on the following subsets: naive, CD62LhighCD44low; and effector, CD62LlowCD44high. Left panel, Ratios for line 2 BAFF Tg mice and WT controls; right panel, ratios for the μMT–/– x BAFF Tg line 1 F2 generation littermates. C, μMT–/– x BAFF Tg mice have similar levels of paw swelling as WT and μMT–/– control mice (n = 6). D, Serum levels of BAFF were measured using ELISA and plotted against paw swelling at 48 h. **, p < 0.01; ***, p < 0.005.
We examined whether disrupted T cell subset ratios in μMT–/– x BAFF Tg mice could affect the course of the DTH reaction. WT, μMT–/–, and μMT–/– x BAFF Tg mice displayed similar levels of footpad swelling over the course of DTH (Fig. 4C). Paw swelling at 48 h was plotted against serum BAFF levels for each animal, and linear regression analysis was performed (Fig. 4D). The correlation coefficient (r2) of <0.01 indicated a lack of correlation between BAFF levels and DTH response in the absence of B cells. Thus, BAFF-mediated changes in B cell numbers or function affect T cell subset composition and responses in the DTH reaction.
BAFF Tg mice have compromised allergic airway responses
We next asked whether the enhanced DTH response in BAFF Tg mice was the result of a general increase in T cell responsiveness, particularly by effector memory T cells, or was related more to a Th1-specific enhancement. The responses of WT and BAFF Tg mice were examined in a Th2-driven OVA model of allergic airway inflammation. Measurement of cell numbers in the BAL fluid showed that exposure of WT mice to OVA aerosol resulted in substantial eosinophil infiltration (Fig. 5A), which is characteristic for this model (41). In contrast, BAFF Tg mice showed a significant reduction in eosinophil infiltration. In accordance with BAL fluid cell numbers, histochemical staining of lung tissue sections revealed greatly reduced numbers of peribronchial and perivascular leukocytes in BAFF Tg mice compared with WT controls (Fig. 5B). Levels of anti-OVA specific IgE Abs in serum were roughly equivalent in WT and BAFF Tg mice (not shown), suggesting that the suppressed airway inflammation in BAFF Tg mice may relate to BAFF effects on T cell function rather than those on B cells or Ab production.
FIGURE 5. Suppression of allergic airway inflammation in BAFF Tg mice. A, BAL fluid was recovered from BAFF Tg mice and WT controls after an 8-day aerosol exposure regimen, and constituent cell types were determined by cytospin and Giemsa staining (n = 5). B, Lung sections stained with H&E reveal greatly reduced leukocyte infiltration in BAFF Tg mice. C, Peribronchial lymph nodes were collected and pooled. Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 100 μg/ml OVA for 72 h, and proliferation was measured by thymidine uptake. D, IL-5 and IFN- levels from culture supernatants were measured by ELISA. ***, p < 0.005.
Lymphocytes from peribronchial lymph nodes were collected after the final aerosol exposure and restimulated in vitro with OVA for 72 h. WT lymphocytes showed a strong increase in proliferation in response to restimulation with OVA, in contrast to lymphocytes from BAFF Tg mice (Fig. 5C). Cytokine measurements showed that after restimulation with OVA, WT lymph nodes cells produced high levels of IL-5, whereas cells from BAFF Tg mice showed 10-fold reduced levels (Fig. 5D). In addition, small, but demonstrable, levels of IFN- were detected in some BAFF Tg cultures, although this result was not always reproducible. Of note, there was no compensatory increase in IL-10 production in BAFF Tg cultures (data not shown). We also examined cytokine levels in BAL fluid, but observed no differences in IL-4 or IL-5 levels between WT and BAFF Tg mice. However, in our experience, BAL fluid measurements for cytokines have not been particularly reliable. In summary, BAFF Tg mice show a suppression of Th2-mediated allergic airway inflammation and an associated reduction in lymph node T cell proliferation and IL-5 production in response to OVA challenge.
Increased OVA-specific recall responses from lymph nodes of BAFF Tg mice
To determine whether initial priming defects were responsible for the observed reduction in airway eosinophilia in BAFF Tg mice, we performed immunization and in vitro recall experiments. Mice were immunized at the tail base with an OVA/alum mix, and inguinal lymph nodes were removed after 7 days and restimulated in vitro for 72 h. As expected, WT lymph node cells showed increased proliferation upon stimulation with OVA. BAFF Tg lymph node cells showed an 3-fold increase in proliferation over WT mice (Fig. 6A). In addition, cytokine ELISA from supernatants of restimulated cultures from BAFF Tg mice demonstrated significantly increased levels of IL-5 and IFN- compared with WT mice (Fig. 6B). Thus, BAFF Tg mice have increased OVA-specific recall responses, and the defect in lung eosinophilia observed in BAFF Tg mice was not due to impaired priming to OVA.
FIGURE 6. Increased OVA-specific in vitro recall responses in BAFF Tg. A, Mice were injected at the tail base with 100 μg of OVA in alum, and inguinal lymph nodes were collected 7 days later (n = 4). Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 100 μg/ml OVA for 72 h. Proliferation was measured by thymidine uptake. B, Cytokine levels from culture supernatants were measured by ELISA. **, p < 0.01.
BAFF-mediated suppression of allergic airway inflammation is B cell independent
Our results demonstrating that μMT–/– x BAFF Tg mice were protected from the augmented DTH responses seen in BAFF Tg mice led to the question of whether BAFF-mediated suppression of airway inflammation would be relieved in μMT–/– x BAFF Tg mice. Differential cell counts from BAL fluid showed no significant differences in cell numbers between WT and μMT–/– mice in response to OVA aerosol, with characteristic eosinophil infiltration observed in either case (Fig. 7A). Similar to BAFF Tg mice, μMT–/– x BAFF Tg mice showed a significant reduction in the eosinophil infiltration in response to OVA challenge. Restimulation with OVA of peribronchial lymph node T cells from WT and μMT–/– mice showed strong induction of proliferation (Fig. 7B) and IL-5 production (Fig. 7C), whereas there was strong suppression of proliferation and IL-5 production in μMT–/– x BAFF Tg mice (Fig. 7, B and C), as seen in BAFF Tg mice. There were no detectable levels of IFN- or IL-10 in any of the cultures (data not shown). These results demonstrate that BAFF-mediated suppression of Th2-dependent allergic airway inflammation occurs by mechanisms that are B cell independent.
FIGURE 7. Suppression of allergic airway inflammation in BAFF Tg mice is B cell independent. A, BAL fluid was recovered from WT, μMT–/–, and μMT–/– x BAFF Tg mice after aerosol exposure, and constituent cell types were determined by cytospin and Giemsa staining (n = 5). B, Peribronchial lymph nodes were collected and pooled into groups. Cultures were normalized to 2 x 105 T cells/well and restimulated with medium alone or 100 μg/ml OVA for 72 h, with proliferation measured by thymidine uptake. C, IL-5 and IFN- levels from culture supernatants were measured by ELISA. *, p < 0.05; ***, p < 0.005.
Discussion
Overproduction of BAFF has been associated with various autoimmune diseases (3, 4, 5, 6, 8, 9). This association was originally thought to be through BAFF effects on B cell maturation and survival. However, it is now clear that BAFF promotes T cell activation (18, 23, 24) and effector function (25, 26), which may contribute to autoimmunity. Autoimmune diseases are often associated with Th1-type T cells and the cytokines they produce, such as IFN- and TNF-. Our results suggest that BAFF is an important cytokine for both B and T cell responses in vivo, and that overproduction of BAFF has profound effects on both the T cell and B cell systems.
Our findings with the DTH model highlight the role for BAFF in modulating T cell responses in vivo. DTH is a classical Th1-mediated response involving Ag-specific T cell activation and production of Th1 cytokines, including TNF- and IFN- (42). BAFF Tg mice developed significantly prolonged DTH responses, and we suggest that this augmented DTH response is due to increased effector T cell expansion, survival, and effector function. Restimulation of lymph node cells supported this conclusion, showing increased T cell proliferation and a 10-fold increase in IFN- production by T cells isolated from BAFF Tg mice. Thus, in a high BAFF environment, T cells receive additional costimulatory signals, expand, and provide augmented effector functions.
Of particular note was the correlation between BAFF levels and the degree of swelling in BAFF Tg mice. BAFF augmented the DTH response in a concentration-dependent manner. However, although BAFF did augment DTH responses, it was not necessary for basal responses. Indeed, BAFF may play only a small role during the course of many normal T cell responses. However, in situations in which there are high serum levels of BAFF, such as in an ongoing autoimmune disease or possibly persistent viral or bacterial infection, there could be increased Th1 T cell activity and increased effector T cell responses. Our data for μMT–/– x BAFF Tg mice showed that B cells were necessary for the dose-dependent effect of BAFF on cutaneous DTH responses. The most straightforward explanation for our results is that high levels of BAFF affect the numbers and phenotype of peripheral B cells (5), which, in turn, affect the makeup of the T cell immune system, particularly the proportion of effector memory T cells. These effector memory T cells, many of which are most probably committed to a Th1-type response, might also receive additional survival or costimulatory signals from the high levels of BAFF present in serum or on the surface of APC.
BAFF augmented the DTH response, a typical Th1 response, suggesting that this cytokine may play a role in immune responses to viral or bacterial infections. BAFF is produced at sites of inflammatory reactions, particularly by leukocyte types associated with type 1 responses, such as neutrophils (43, 44) and macrophages (45). To date, most studies have been confined to autoimmune diseases, with few addressing BAFF levels after viral or bacterial infection due partly to the lack of commercial reagents. In HIV-1 infection, BAFF levels were elevated compared with control values, particularly during later stages of infection when CD4 counts had declined (46). In addition, HIV-1-infected patients show alterations in their B cell immune system consistent with high BAFF levels, notably hypergammaglobulinemia and altered B cell differentiation (47). BAFF produced at an inflammatory site by activated neutrophils, activated T cells, or macrophages would be transported via the lymph to local lymph nodes, augment T cell activation, and probably provide survival signals to the central and effector memory T cells that express BAFF-R (18). In BAFFhigh individuals, increased proportions of effector memory T cells, possibly predisposed to become Th1-type effector T cells, would promote the clearance of viral or bacterial agents.
BAFF Tg mice had suppressed allergic airway responses, with a marked reduction in eosinophils in BAL fluid and infiltrating leukocytes around airways and pulmonary blood vessels. Restimulation of lung-draining lymph node cells with OVA showed markedly reduced T cell proliferation and IL-5 production in BAFF Tg mice. This would account for the reduced eosinophils in the airways, because IL-5 is the key cytokine regulating eosinophil production and recruitment in this model (48). Presumably this suppression of Th2 effector function was the result of skewing of the OVA-specific T cell response toward that of a more Th1-like profile. There was some evidence for this, because small amounts of IFN- were produced by T cells in lung-draining lymph nodes from BAFF Tg mice, and this was not observed in WT mice. However, other explanations exist, including local regulatory T cells that may be particularly BAFF responsive and associated with airway tissue and its draining lymph nodes. Such regulatory T cells have been shown to inhibit Th2 airway responses in mice (49). However, at the whole animal level, BAFF Tg mice were effectively primed to OVA, because restimulation of non-lung-draining (inguinal) lymph node T cells from primed BAFF Tg mice revealed significantly increased proliferation and IL-5 and IFN- production, similar to the enhanced recall response seen in mBSA-primed BAFF Tg mice. It is also possible that an increase in IFN- production at the initial stages of priming may lead to antagonism of Th2 differentiation. IFN- induces the Th1 transcription factor T-bet, which can suppress IL-5 production (50). However, the very localized suppression of T cell responses in lung-associated lymph nodes, but not other lymph nodes, might favor the notion of a pulmonary Th1-like regulatory T cell, as has been described in a recent study (51). These T-bet- and forkhead/winged helix transcription factor gene-expressing regulatory T cells suppressed the development of airway hyper-reactivity, possibly through the actions of IL-10 (51). However, we have found no strong evidence for a suppressive IL-10-mediated T cell response in BAFF Tg lung-associated lymph nodes and no changes in CD4+CD25+ regulatory T cells or increased levels of IL-10 (data not shown). Interestingly, a large proportion of CD4+CD25+ regulatory T cells in the mouse express BAFF-R (26). At present, T and B cells are the only cell types with strong evidence of BAFF-R expression, so the mechanism presumably relates to BAFF signaling to either a T or B cell subset. However, the inhibition by BAFF of allergic airway responses in μMT–/– x BAFF Tg mice appears to exclude B cell involvement. One important qualification that must be noted is that the enhanced DTH response in BAFF Tg mice was in skin, whereas the suppressed response in the allergic model was in mucosal tissues. It is conceivable that BAFF effects somehow relate to the nature of cutaneous or mucosal responses.
In conclusion, we show that BAFF overexpression in vivo promotes the DTH reaction, which is a classical Th1 response, and suppresses Th2-dependent allergic airway responses. The effects of BAFF on the T cell system were profound in terms of subset ratios and Th1 or Th2 responses. Whether BAFFhigh human subjects also show augmented Th1-type responses or suppressed Th2 responses will be an interesting topic for future studies.
Acknowledgments
We thank Ian Mackay, Kate Jeffrey, and Robert Sutherland for helpful suggestions. BAFF Tg and BAFF KO mice were supplied by Biogen-Idec, Inc. We also thank Eric Schmied and the staff at the Biological Testing Facility for assistance with animal care.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Australian National Health and Medical Research Council, the Wellcome Trust, and the Cooperative Research Center for Asthma.
2 A.P.R.S. and L.G.N. contributed equally.
3 Address correspondence and reprint requests to Dr. Charles R. Mackay, Arthritis and Inflammation Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail address: c.mackay@garvan.org.au
4 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; BAFF-R, BAFF receptor; BAL, bronchoalveolar lavage; DTH, delayed-type hypersensitivity; mBSA, methylated BSA; Tg, transgenic; WT, wild type; TACI, transmembrane activator and calcium modulator cyclophilin ligand interactor; BCMA, B cell maturation Ag.
Received for publication December 13, 2004. Accepted for publication February 14, 2005.
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B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.
Introduction
Bcell-activating factor belonging to the TNF family (BAFF; 4 BLyS, TALL-1, zTNF-4, THANK) is a TNF superfamily member that plays an important role in immune responses (1, 2). Excessive production of BAFF is associated with the development of autoimmune diseases, because transgenic (Tg) mice that overproduce BAFF develop severe autoimmune disorders that resemble systemic lupus erythematosus and Sj?gren’s syndrome (3, 4, 5, 6). Also, high levels of BAFF have been found in the blood of patients with autoimmune diseases, particularly systemic lupus erythematosus and Sj?gren’s syndrome (5, 7, 8, 9). Thus, excess BAFF may cause a breakdown in immune tolerance through inappropriate survival signals to splenic and peripheral B cells (10, 11, 12). In the spleen, BAFF promotes B cell maturation, particularly at the critical T1-T2 immune tolerance checkpoint (10), and splenic B cells in BAFF-deficient mice fail to mature past the T1 stage (13). Autoreactive B cells are expanded in lymphoid tissues of BAFF Tg mice (3, 14), possibly as a result of BAFF rescue of autoreactive cells that would normally be deleted (12, 14). BAFF also enhances the survival of plasmablasts (15, 16) and stimulates peripheral B cell proliferation, Ab production, and class switching (17, 18, 19).
BAFF binds three receptors: BAFF-R, transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA) (1). BAFF-R is the predominant BAFF receptor on circulating B cells (18) and is also important for the regulation of B cell maturation in the spleen, because BAFF-R-deficient mice show disrupted B cell maturation, similar to that seen in BAFF-deficient mice (13, 20). BAFF-induced survival is mediated by up-regulation of the antiapoptotic molecule Bcl-2 (3), and Bcl-2 overexpression restores B cell survival in the absence of BAFF (21). The other receptors, TACI and BCMA, have restricted patterns of expression on specific B cell subsets (18); BCMA appears to be particularly important for survival or stimulation of plasmablasts and germinal center cells (15, 18), whereas TACI most likely serves as a negative regulator of B cell activity, particularly on activated B cells (22).
More recently, BAFF has emerged as a regulator of T cell function. BAFF provides costimulatory signals to T cells in the presence of suboptimal TCR stimulation and enhances T cell proliferation and cytokine production (18, 23, 24). TACI-Ig and BAFF-R-Ig decoy receptors inhibit T cell activation in vitro (18, 23) and in vivo (25), and BAFF-deficient mice show impaired T cell-mediated allograft rejection (26). Thus, physiological levels of BAFF appear necessary for the generation of optimal T cell responses. These effects occur through BAFF-R, because T cells from BAFF-R mutant mice fail to respond to BAFF, and BAFF-R expression (but not that of TACI or BCMA) is present on the surface of subsets of central and effector memory T cells (18). These findings suggest that BAFF may play a role in stimulating T cell function in vivo, particularly during T cell-mediated pathogenic reactions. Interestingly, BAFF is highly up-regulated in astrocytes in multiple sclerosis plaques (27) and is also produced by fibroblast-like synoviocytes from rheumatoid arthritis patients (28). Both of these autoimmune diseases have a strong T cell association.
CD4+ effector T cells often polarize to either Th1 or Th2 phenotypes (29). Th1 responses control intracellular pathogens such as viruses and are associated with the production of IFN-, IL-2, and TNF- and recruitment of phagocytic leukocytes. In contrast, Th2 responses control infections by large extracellular parasites, in part through production of IL-4, IL-5, and IL-13, and recruitment of eosinophils. Dysregulation of Th1 or Th2 responses may contribute to the pathogenesis of autoimmune diseases as well as allergic diseases such as asthma. The mechanisms that initiate and maintain polarization of T cell responses to Th1 or Th2 are incompletely understood, but instructive signals in the form of cytokines and cell surface molecules expressed by APC are important (30, 31, 32, 33). Th1 and Th2 responses counteract each other, in that strong polarization to a Th1 response suppresses Th2 responses and vice versa (34). Given the ability of BAFF to regulate T cell activation and its association with autoimmune diseases, we investigated whether BAFF affects the outcome of Th1- and Th2-mediated T cell responses in vivo.
We show that systemic overexpression of BAFF in BAFF Tg mice exacerbates the severity of Th1-mediated delayed-type hypersensitivity (DTH) responses by enhanced T cell proliferation and IFN- production in local lymph nodes. Enhanced DTH responses in BAFF Tg mice correlated directly with levels of BAFF in serum, suggesting that the varying levels of BAFF seen in the blood of human subjects might also relate to varying capacities for DTH Th1 responses. High levels of BAFF in BAFF Tg mice also resulted in subdued Th2-mediated responses in an allergic airways disease model. The effects of BAFF on Th1/Th2 responses operated at multiple levels, because enhanced Th1 responses depended on B cells, whereas BAFF inhibition of Th2 responses was B cell independent. These findings demonstrate that BAFF augments certain Th1 responses in vivo, but not Th2 responses, and illustrate the importance of BAFF for T cell as well as B cell responses.
Materials and Methods
Reagents and Abs
Methylated BSA (mBSA) and OVA were purchased from Sigma-Aldrich. Imject CFA and Imject alum were purchased from Pierce. All anti-mouse FITC-, PE-, CyChrome-, PerCP-, and allophycocyanin-conjugated mAbs were obtained from BD Pharmingen. Murine IL-4, IL-5, IFN-, and IL-10 ELISA kits were purchased from BD Biosciences. Goat anti-mouse alkaline phosphatase-conjugated Abs were purchased from Southern Biotechnology Associates. Human BCMA-Ig was provided by Biogen-Idec and has been described previously (35).
Mice
Animals were housed under conventional barrier protection and handled in accordance with the animal experimentation ethics committee, which complies with the Australian code of practice for the care and use of animals for scientific purposes. BAFF Tg mice (3) and BAFF–/– mice (13) were supplied by Biogen-Idec and were bred in our animal facility. BAFF Tg mice were maintained as mice homozygous for the BAFF transgene, and wild-type (WT) mice were used as controls, as previously described (36). Two lines of BAFF Tg mice were used for experimentation, referred to as lines 1 and 2, which differ slightly in BAFF levels and immune responses (see below). μMT–/– mice (37) were purchased from The Jackson Laboratory. μMT–/– x BAFF Tg mice were established by breeding homozygous μMT–/– and BAFF Tg line 1 mice and subsequent breeding of the F1 generation. Homozygous μMT–/– x BAFF Tg mice and WT controls were derived from the F2 generation and bred as separate lines for experimentation. All mice were maintained on a C57BL/6 background
DTH responses
Six- to 8-wk-old mice were injected s.c. at the tail base with 200 μl of 1.25 mg/ml mBSA in CFA (1/1 mix) on day 1, as described previously (38). On day 7, mice were rechallenged in one hind footpad with an injection of 20 μl of a 10 mg/ml mBSA solution, whereas the opposite hind footpad received 20 μl of 1x PBS. Paw swelling was measured 8–72 h after rechallenge with a dial thickness gauge. For paw tissue section analysis, mice were killed at 48 h after challenge. The paws of mice were dissected and fixed in 10% formalin (Sigma-Aldrich) in PBS for 5 days. Paws were decalcified for 10% formalin in PBS supplemented with formic acid (Sigma-Aldrich) at a 9:1 ratio before being embedded in paraffin, sectioned, and stained with H&E.
For analysis of mBSA-specific Abs, mice were killed 7 days after footpad challenge, serum was collected, and mBSA-specific Abs levels were measured by ELISA. In vitro recall responses were measured by collecting inguinal lymph nodes 7 days after immunization. Cultures were normalized to 2 x 106 T cells/ml and were restimulated for 72 h with 40 μg/ml mBSA in X-Vivo 15 serum-free medium (BioWhittaker) supplemented with penicillin/streptomycin. Supernatants were collected for measurement of cytokine production by ELISA. Proliferation was measured by addition of 1 μCi of [3H]thymidine (Amersham Biosciences)/well 18 h before harvesting and subsequent beta scintillation counting.
OVA-induced allergic airway inflammation
Six- to 8-wk-old mice were injected i.p. with 200 μl of a 1 mg/ml OVA solution with alum (1/1 mix) on days 1 and 15. Mice were given a 20-min aerosol on days 28, 30, 32, and 34 consisting of either PBS or 1% OVA in PBS. Mice were killed on day 35, and bronchoalveolar lavage (BAL) fluid and organs were collected. Total BAL fluid cell numbers were enumerated, and differential cell counts were performed after cytospin and Giemsa staining to establish the number and identity of infiltrating cells. Lung tissues were fixed in 10% Formalin/PBS for 7 days and embedded in paraffin. Standard protocols for H&E staining were used to stain tissue sections. At the completion of the aerosol regimen, peribronchial lymph nodes were collected, pooled into groups, and cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS and penicillin/streptomycin. Cultures were normalized to 2 x 106 T cells/ml and restimulated for 72 h with 100 μg/ml OVA, and proliferation and cytokine production were measured as per DTH. In vitro recall responses were measured by immunizing mice at the tail base with 200 μl of 1 mg/ml OVA in alum (1/1 mix) and collecting inguinal lymph nodes 7 days later. Cells were restimulated in vitro as described above for peribronchial lymph nodes, using 500 μg/ml OVA.
ELISA
The mouse BAFF ELISA was performed as previously described (36). The Ab response to mBSA was analyzed as follows. MaxiSorb plates (Nalge Nunc International; 384-well) were coated with 50 μg/ml mBSA diluted in 0.1 M sodium bicarbonate buffer (pH 9.6) at 4°C overnight. Plates were washed three times with PBS-0.05% Tween 20. Serial dilutions of mouse serum in ELISA buffer (1% BSA in PBS) were added to the plate. Anti-mBSA Abs were detected using 0.125 μg/ml alkaline phosphatase-conjugated goat anti-mouse IgM, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, or anti-IgA (Southern Biotechnology Associates). p-Nitrophenyl phosphate tablets (Sigma-Aldrich) were used for detection, and plates were read at an OD of 405 nm. The titer (log base 2) is defined as the serum dilution giving an OD four times higher than that of background (where 1 = 1/50 dilution).
Statistical analysis
Statistical significance was determined using t test, and significance with relation to comparison data is indicated by p values in the figure legends.
Results
Enhanced DTH responses in BAFF Tg mice
Six- to 8-wk-old mice were immunized intradermally at the base of the tail with mBSA in CFA and challenged 7 days later with an s.c. injection of mBSA into the footpad. BAFF Tg mice from two distinct transgenic lines (lines 1 and 2) displayed significantly increased paw swelling compared with WT mice (Fig. 1, A and B). Paw swelling was comparable in WT and BAFF Tg mice at early time points after challenge (Fig. 1A), but was significantly higher in BAFF Tg mice 48 and 72 h after challenge. DTH responses in BAFF Tg mice at 48 h were also characterized by increased erythema (Fig. 1B), and histological analysis of paw tissue sections revealed an increased cellular infiltrate into the footpads (Fig. 1C). Serum anti-mBSA Ab isotypes measured 7 days after footpad challenge showed BAFF Tg mice to have significantly higher titers of mBSA-specific IgG1, IgG2a, and IgG2b compared with WT mice (Fig. 1D), whereas mBSA-specific IgA levels showed a nonsignificant trend toward an increase (data not shown). Levels of mBSA-specific IgM and IgG3 were similar in WT and BAFF Tg mice (data not shown).
FIGURE 1. BAFF Tg mice show increased DTH responses. A, Increased paw swelling over time in two lines of BAFF Tg mice (n = 10, line 1; n = 6, line 2). B, Increased erythema in paws of BAFF Tg mice at 48 h compared with WT mice. C, H&E staining of paw sections at 48 h reveals increased leukocyte infiltration in BAFF Tg mice compared with WT controls. D, Increased serum levels of mBSA-specific Abs in BAFF Tg mice 7 days after footpad challenge (n = 11, WT; n = 10, BAFF Tg). *, p < 0.05; **, p < 0.01; ***, p < 0.005.
BAFF levels determine the magnitude of the DTH response
The concentration of BAFF in human blood varies considerably; most individuals express low levels, but some autoimmune patients express levels >100 ng/ml (5). BAFF Tg mice also show variable levels of BAFF in blood due to secondary endogenous BAFF production in response to autoimmune activation (36). To assess the relationship between serum BAFF levels and paw swelling, serum from both WT and BAFF Tg mice was collected 48 h after challenge, and the level of BAFF in serum was determined by ELISA. Both lines of BAFF Tg mice displayed high levels of BAFF in serum (line 1 average concentration, 215.03 ± 50.69 ng/ml; line 2 average concentration, 1128.2 ± 315.8 ng/ml), whereas lower levels of BAFF were detected in WT mice (5.27 ± 1.47 ng/ml), similar to levels in unimmunized mice (36). When the level of BAFF in serum was correlated with paw swelling for each mouse (Fig. 2A), a strong correlation was observed (r2 = 0.593). Thus systemic BAFF levels may regulate the magnitude of certain T cell responses.
FIGURE 2. DTH responses correlate with serum BAFF levels, although BAFF deficiency does not eliminate DTH responses. A, Serum levels of BAFF in BAFF Tg mice were measured using ELISA and plotted against paw swelling 48 h after footpad rechallenge. B, DTH responses in BAFF–/– mice, and mice heterozygous for the knockout allele (n = 6). C, C57/B6 mice treated with BCMA-Ig showed no reduction in DTH severity compared with control (PBS-treated) mice (n = 6).
We next examined DTH responses in BAFF-deficient mice. These mice were competent in mounting a normal DTH response (Fig. 2B). Also, WT mice treated with BCMA-Ig showed no consistent reduction in DTH responses compared with untreated mice (Fig. 2C). Thus, BAFF is not essential for basal DTH responses, but in circumstances of BAFF overproduction, the magnitude of DTH responses correlates with the level of BAFF.
Increased mBSA-specific T cell proliferation and IFN- production in BAFF Tg mice
The mechanisms underlying the enhanced DTH response in BAFF Tg mice were investigated, first by examining T cell responses to mBSA in vitro. WT and BAFF Tg mice were immunized at the tail base as described above, and inguinal lymph nodes were collected 7 days later. Lymph node cells were restimulated in vitro with mBSA for 72 h. T cells from BAFF Tg mice gave a significantly stronger recall response compared with WT mice, displaying a 3-fold increase in proliferation (Fig. 3A) and a 10-fold higher IFN- production in culture supernatants (Fig. 3B). IL-4 and IL-5 levels were below the level of detection in both WT and BAFF Tg mice (data not shown). Thus, in an environment of high BAFF levels, T cell responses to Ag are augmented, and this might be due to qualitative changes in the T cells involved or an increased frequency of Ag-specific memory or effector T cells.
FIGURE 3. Increased mBSA-specific in vitro recall responses by T cells in BAFF Tg mice. A, Mice were injected at the tail base with 250 μg of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 40 μg/ml mBSA for 72 h. Proliferation was measured by thymidine uptake. B, IFN- levels from culture supernatants were measured by ELISA (n = 3). *, p < 0.05.
Numbers of effector memory T cells are increased in BAFF Tg mice, but not in μMT–/– x BAFF Tg mice
We also examined changes in the make-up of the T cell immune system that result from long term exposure to high levels of BAFF and effects that expanded subsets of B cells could have on T cell and DTH responses in BAFF Tg mice. Previous studies of BAFF Tg mice revealed altered CD4+ T cell subset ratios (3). Splenocytes from WT and BAFF Tg mice were isolated and stained with Abs to CD4, CD44, and CD62L. We observed a substantial increase in the proportion of CD44highCD62Llow effector memory CD4+ cells and a decrease in CD44lowCD62Lhigh naive CD4+ T cells in BAFF Tg mice (Fig. 4A, top panels). Functionally, effector memory T cells are closely related to effector T cells and have the capacity to migrate to peripheral tissues (39, 40). To investigate whether the expansion of mature B cell subsets seen in BAFF Tg mice (3) might in some way connect to the increased numbers of effector memory CD4+ T cells, we generated B cell-deficient BAFF Tg (μMT–/– x BAFF Tg) mice, and these showed no increase in effector memory T cell numbers (Fig. 4A, bottom panels). The ratio of effector/naive T cells in line 2 BAFF Tg mice was increased 4-fold (Fig. 4B, left panel), and line 1 BAFF Tg mice also showed a statistically significant (although smaller) increase (Fig. 4B, right panel). Analysis of μMT–/– mice showed a reduced ratio, indicating an overall reduction in effector cells, which was not increased in μMT–/– x BAFF Tg mice (line 1). Thus, the altered makeup of the T cell compartment in BAFF Tg mice is dependent on B cells.
FIGURE 4. Altered T cell phenotype and increased DTH severity in BAFF Tg mice are B cell dependent. A, Splenocytes were stained with anti-CD4, anti-CD44, and anti-CD62L (CD4+ gated cells are shown; n = 3 for all genotypes with a representative plot for each genotype shown). B, Cell ratios for CD4+ T cells calculated based on the following subsets: naive, CD62LhighCD44low; and effector, CD62LlowCD44high. Left panel, Ratios for line 2 BAFF Tg mice and WT controls; right panel, ratios for the μMT–/– x BAFF Tg line 1 F2 generation littermates. C, μMT–/– x BAFF Tg mice have similar levels of paw swelling as WT and μMT–/– control mice (n = 6). D, Serum levels of BAFF were measured using ELISA and plotted against paw swelling at 48 h. **, p < 0.01; ***, p < 0.005.
We examined whether disrupted T cell subset ratios in μMT–/– x BAFF Tg mice could affect the course of the DTH reaction. WT, μMT–/–, and μMT–/– x BAFF Tg mice displayed similar levels of footpad swelling over the course of DTH (Fig. 4C). Paw swelling at 48 h was plotted against serum BAFF levels for each animal, and linear regression analysis was performed (Fig. 4D). The correlation coefficient (r2) of <0.01 indicated a lack of correlation between BAFF levels and DTH response in the absence of B cells. Thus, BAFF-mediated changes in B cell numbers or function affect T cell subset composition and responses in the DTH reaction.
BAFF Tg mice have compromised allergic airway responses
We next asked whether the enhanced DTH response in BAFF Tg mice was the result of a general increase in T cell responsiveness, particularly by effector memory T cells, or was related more to a Th1-specific enhancement. The responses of WT and BAFF Tg mice were examined in a Th2-driven OVA model of allergic airway inflammation. Measurement of cell numbers in the BAL fluid showed that exposure of WT mice to OVA aerosol resulted in substantial eosinophil infiltration (Fig. 5A), which is characteristic for this model (41). In contrast, BAFF Tg mice showed a significant reduction in eosinophil infiltration. In accordance with BAL fluid cell numbers, histochemical staining of lung tissue sections revealed greatly reduced numbers of peribronchial and perivascular leukocytes in BAFF Tg mice compared with WT controls (Fig. 5B). Levels of anti-OVA specific IgE Abs in serum were roughly equivalent in WT and BAFF Tg mice (not shown), suggesting that the suppressed airway inflammation in BAFF Tg mice may relate to BAFF effects on T cell function rather than those on B cells or Ab production.
FIGURE 5. Suppression of allergic airway inflammation in BAFF Tg mice. A, BAL fluid was recovered from BAFF Tg mice and WT controls after an 8-day aerosol exposure regimen, and constituent cell types were determined by cytospin and Giemsa staining (n = 5). B, Lung sections stained with H&E reveal greatly reduced leukocyte infiltration in BAFF Tg mice. C, Peribronchial lymph nodes were collected and pooled. Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 100 μg/ml OVA for 72 h, and proliferation was measured by thymidine uptake. D, IL-5 and IFN- levels from culture supernatants were measured by ELISA. ***, p < 0.005.
Lymphocytes from peribronchial lymph nodes were collected after the final aerosol exposure and restimulated in vitro with OVA for 72 h. WT lymphocytes showed a strong increase in proliferation in response to restimulation with OVA, in contrast to lymphocytes from BAFF Tg mice (Fig. 5C). Cytokine measurements showed that after restimulation with OVA, WT lymph nodes cells produced high levels of IL-5, whereas cells from BAFF Tg mice showed 10-fold reduced levels (Fig. 5D). In addition, small, but demonstrable, levels of IFN- were detected in some BAFF Tg cultures, although this result was not always reproducible. Of note, there was no compensatory increase in IL-10 production in BAFF Tg cultures (data not shown). We also examined cytokine levels in BAL fluid, but observed no differences in IL-4 or IL-5 levels between WT and BAFF Tg mice. However, in our experience, BAL fluid measurements for cytokines have not been particularly reliable. In summary, BAFF Tg mice show a suppression of Th2-mediated allergic airway inflammation and an associated reduction in lymph node T cell proliferation and IL-5 production in response to OVA challenge.
Increased OVA-specific recall responses from lymph nodes of BAFF Tg mice
To determine whether initial priming defects were responsible for the observed reduction in airway eosinophilia in BAFF Tg mice, we performed immunization and in vitro recall experiments. Mice were immunized at the tail base with an OVA/alum mix, and inguinal lymph nodes were removed after 7 days and restimulated in vitro for 72 h. As expected, WT lymph node cells showed increased proliferation upon stimulation with OVA. BAFF Tg lymph node cells showed an 3-fold increase in proliferation over WT mice (Fig. 6A). In addition, cytokine ELISA from supernatants of restimulated cultures from BAFF Tg mice demonstrated significantly increased levels of IL-5 and IFN- compared with WT mice (Fig. 6B). Thus, BAFF Tg mice have increased OVA-specific recall responses, and the defect in lung eosinophilia observed in BAFF Tg mice was not due to impaired priming to OVA.
FIGURE 6. Increased OVA-specific in vitro recall responses in BAFF Tg. A, Mice were injected at the tail base with 100 μg of OVA in alum, and inguinal lymph nodes were collected 7 days later (n = 4). Cultures were normalized to 2 x 105 T cells/well and were cultured in triplicate with medium alone or 100 μg/ml OVA for 72 h. Proliferation was measured by thymidine uptake. B, Cytokine levels from culture supernatants were measured by ELISA. **, p < 0.01.
BAFF-mediated suppression of allergic airway inflammation is B cell independent
Our results demonstrating that μMT–/– x BAFF Tg mice were protected from the augmented DTH responses seen in BAFF Tg mice led to the question of whether BAFF-mediated suppression of airway inflammation would be relieved in μMT–/– x BAFF Tg mice. Differential cell counts from BAL fluid showed no significant differences in cell numbers between WT and μMT–/– mice in response to OVA aerosol, with characteristic eosinophil infiltration observed in either case (Fig. 7A). Similar to BAFF Tg mice, μMT–/– x BAFF Tg mice showed a significant reduction in the eosinophil infiltration in response to OVA challenge. Restimulation with OVA of peribronchial lymph node T cells from WT and μMT–/– mice showed strong induction of proliferation (Fig. 7B) and IL-5 production (Fig. 7C), whereas there was strong suppression of proliferation and IL-5 production in μMT–/– x BAFF Tg mice (Fig. 7, B and C), as seen in BAFF Tg mice. There were no detectable levels of IFN- or IL-10 in any of the cultures (data not shown). These results demonstrate that BAFF-mediated suppression of Th2-dependent allergic airway inflammation occurs by mechanisms that are B cell independent.
FIGURE 7. Suppression of allergic airway inflammation in BAFF Tg mice is B cell independent. A, BAL fluid was recovered from WT, μMT–/–, and μMT–/– x BAFF Tg mice after aerosol exposure, and constituent cell types were determined by cytospin and Giemsa staining (n = 5). B, Peribronchial lymph nodes were collected and pooled into groups. Cultures were normalized to 2 x 105 T cells/well and restimulated with medium alone or 100 μg/ml OVA for 72 h, with proliferation measured by thymidine uptake. C, IL-5 and IFN- levels from culture supernatants were measured by ELISA. *, p < 0.05; ***, p < 0.005.
Discussion
Overproduction of BAFF has been associated with various autoimmune diseases (3, 4, 5, 6, 8, 9). This association was originally thought to be through BAFF effects on B cell maturation and survival. However, it is now clear that BAFF promotes T cell activation (18, 23, 24) and effector function (25, 26), which may contribute to autoimmunity. Autoimmune diseases are often associated with Th1-type T cells and the cytokines they produce, such as IFN- and TNF-. Our results suggest that BAFF is an important cytokine for both B and T cell responses in vivo, and that overproduction of BAFF has profound effects on both the T cell and B cell systems.
Our findings with the DTH model highlight the role for BAFF in modulating T cell responses in vivo. DTH is a classical Th1-mediated response involving Ag-specific T cell activation and production of Th1 cytokines, including TNF- and IFN- (42). BAFF Tg mice developed significantly prolonged DTH responses, and we suggest that this augmented DTH response is due to increased effector T cell expansion, survival, and effector function. Restimulation of lymph node cells supported this conclusion, showing increased T cell proliferation and a 10-fold increase in IFN- production by T cells isolated from BAFF Tg mice. Thus, in a high BAFF environment, T cells receive additional costimulatory signals, expand, and provide augmented effector functions.
Of particular note was the correlation between BAFF levels and the degree of swelling in BAFF Tg mice. BAFF augmented the DTH response in a concentration-dependent manner. However, although BAFF did augment DTH responses, it was not necessary for basal responses. Indeed, BAFF may play only a small role during the course of many normal T cell responses. However, in situations in which there are high serum levels of BAFF, such as in an ongoing autoimmune disease or possibly persistent viral or bacterial infection, there could be increased Th1 T cell activity and increased effector T cell responses. Our data for μMT–/– x BAFF Tg mice showed that B cells were necessary for the dose-dependent effect of BAFF on cutaneous DTH responses. The most straightforward explanation for our results is that high levels of BAFF affect the numbers and phenotype of peripheral B cells (5), which, in turn, affect the makeup of the T cell immune system, particularly the proportion of effector memory T cells. These effector memory T cells, many of which are most probably committed to a Th1-type response, might also receive additional survival or costimulatory signals from the high levels of BAFF present in serum or on the surface of APC.
BAFF augmented the DTH response, a typical Th1 response, suggesting that this cytokine may play a role in immune responses to viral or bacterial infections. BAFF is produced at sites of inflammatory reactions, particularly by leukocyte types associated with type 1 responses, such as neutrophils (43, 44) and macrophages (45). To date, most studies have been confined to autoimmune diseases, with few addressing BAFF levels after viral or bacterial infection due partly to the lack of commercial reagents. In HIV-1 infection, BAFF levels were elevated compared with control values, particularly during later stages of infection when CD4 counts had declined (46). In addition, HIV-1-infected patients show alterations in their B cell immune system consistent with high BAFF levels, notably hypergammaglobulinemia and altered B cell differentiation (47). BAFF produced at an inflammatory site by activated neutrophils, activated T cells, or macrophages would be transported via the lymph to local lymph nodes, augment T cell activation, and probably provide survival signals to the central and effector memory T cells that express BAFF-R (18). In BAFFhigh individuals, increased proportions of effector memory T cells, possibly predisposed to become Th1-type effector T cells, would promote the clearance of viral or bacterial agents.
BAFF Tg mice had suppressed allergic airway responses, with a marked reduction in eosinophils in BAL fluid and infiltrating leukocytes around airways and pulmonary blood vessels. Restimulation of lung-draining lymph node cells with OVA showed markedly reduced T cell proliferation and IL-5 production in BAFF Tg mice. This would account for the reduced eosinophils in the airways, because IL-5 is the key cytokine regulating eosinophil production and recruitment in this model (48). Presumably this suppression of Th2 effector function was the result of skewing of the OVA-specific T cell response toward that of a more Th1-like profile. There was some evidence for this, because small amounts of IFN- were produced by T cells in lung-draining lymph nodes from BAFF Tg mice, and this was not observed in WT mice. However, other explanations exist, including local regulatory T cells that may be particularly BAFF responsive and associated with airway tissue and its draining lymph nodes. Such regulatory T cells have been shown to inhibit Th2 airway responses in mice (49). However, at the whole animal level, BAFF Tg mice were effectively primed to OVA, because restimulation of non-lung-draining (inguinal) lymph node T cells from primed BAFF Tg mice revealed significantly increased proliferation and IL-5 and IFN- production, similar to the enhanced recall response seen in mBSA-primed BAFF Tg mice. It is also possible that an increase in IFN- production at the initial stages of priming may lead to antagonism of Th2 differentiation. IFN- induces the Th1 transcription factor T-bet, which can suppress IL-5 production (50). However, the very localized suppression of T cell responses in lung-associated lymph nodes, but not other lymph nodes, might favor the notion of a pulmonary Th1-like regulatory T cell, as has been described in a recent study (51). These T-bet- and forkhead/winged helix transcription factor gene-expressing regulatory T cells suppressed the development of airway hyper-reactivity, possibly through the actions of IL-10 (51). However, we have found no strong evidence for a suppressive IL-10-mediated T cell response in BAFF Tg lung-associated lymph nodes and no changes in CD4+CD25+ regulatory T cells or increased levels of IL-10 (data not shown). Interestingly, a large proportion of CD4+CD25+ regulatory T cells in the mouse express BAFF-R (26). At present, T and B cells are the only cell types with strong evidence of BAFF-R expression, so the mechanism presumably relates to BAFF signaling to either a T or B cell subset. However, the inhibition by BAFF of allergic airway responses in μMT–/– x BAFF Tg mice appears to exclude B cell involvement. One important qualification that must be noted is that the enhanced DTH response in BAFF Tg mice was in skin, whereas the suppressed response in the allergic model was in mucosal tissues. It is conceivable that BAFF effects somehow relate to the nature of cutaneous or mucosal responses.
In conclusion, we show that BAFF overexpression in vivo promotes the DTH reaction, which is a classical Th1 response, and suppresses Th2-dependent allergic airway responses. The effects of BAFF on the T cell system were profound in terms of subset ratios and Th1 or Th2 responses. Whether BAFFhigh human subjects also show augmented Th1-type responses or suppressed Th2 responses will be an interesting topic for future studies.
Acknowledgments
We thank Ian Mackay, Kate Jeffrey, and Robert Sutherland for helpful suggestions. BAFF Tg and BAFF KO mice were supplied by Biogen-Idec, Inc. We also thank Eric Schmied and the staff at the Biological Testing Facility for assistance with animal care.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Australian National Health and Medical Research Council, the Wellcome Trust, and the Cooperative Research Center for Asthma.
2 A.P.R.S. and L.G.N. contributed equally.
3 Address correspondence and reprint requests to Dr. Charles R. Mackay, Arthritis and Inflammation Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail address: c.mackay@garvan.org.au
4 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; BAFF-R, BAFF receptor; BAL, bronchoalveolar lavage; DTH, delayed-type hypersensitivity; mBSA, methylated BSA; Tg, transgenic; WT, wild type; TACI, transmembrane activator and calcium modulator cyclophilin ligand interactor; BCMA, B cell maturation Ag.
Received for publication December 13, 2004. Accepted for publication February 14, 2005.
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