CD19 regulates the development of bleomycin-induced pulmonary fibrosis in a mouse model

Authors


Abstract

Objective

The contribution of CD19 and B lymphocytes to pulmonary fibrosis is controversial. The aim of this study was to address the role of CD19 during the development of pulmonary fibrosis.

Methods

Mice lacking or overexpressing the B cell surface molecule CD19, which is known as a positive regulator of B cell activation, were used in a model of bleomycin-induced pulmonary fibrosis. Ten or sixteen days after intratracheal injection of bleomycin, lung sections from mice were evaluated by histologic analysis. Seven days after instillation, the total leukocyte count and the number of B cells in bronchoalveolar lavage fluid (BALF) were determined, using a hemocytometer and flow cytometry. Bleomycin was also administered into selectin-deficient or intercellular adhesion molecule 1–deficient mouse strains. The level of CXCR3 expression on B cells was determined by flow cytometry.

Results

CD19 deficiency significantly reduced susceptibility to intratracheal bleomycin challenge on day 16, while CD19 overexpression augmented fibrosis even on day 10. Furthermore, the survival rate and number of B cells in BALF also correlated with CD19 expression levels. The accumulation of B cells in BALF was dependent on CD19 levels, whereas there was no association with the levels of selectins or intercellular adhesion molecule 1. Additionally, CXCR3 was up-regulated in BALF B cells, while it was rarely expressed on circulating B cells. Furthermore, CD19 signaling facilitated B cell CXCR3 up-regulation in response to stimulation in vitro.

Conclusion

These results suggest that CD19 signaling is associated with the development of pulmonary fibrosis by controlling B cell infiltration into lung tissue, which may be associated with CXCR3 up-regulation.

Pulmonary fibrosis comprises a diverse group of diseases characterized by inflammatory infiltrates, disruption of the alveolar structure, and excessive synthesis and deposition of connective tissue (1). Idiopathic pulmonary fibrosis is a fatal disorder with a 5-year survival rate of ∼50% (2). Pulmonary fibrosis is frequently associated with certain autoimmune disorders, including systemic sclerosis and inflammatory muscle diseases. Although the pathogenesis of pulmonary fibrosis remains unknown, it has been treated using immunosuppressive agents.

To examine the underlying pathophysiology, bleomycin-induced lung injury is widely used as an animal model for pulmonary fibrosis (1, 3). Intratracheal administration of bleomycin induces acute alveolar injury, accompanied by the recruitment of leukocytes in the first week and fibrotic responses in the second week (4, 5). In a hamster model, pulmonary fibrosis decreased after 4 weeks of treatment with bleomycin (6). Therefore, leukocytes infiltrating into the lung are likely to play a critical role in pulmonary fibrosis by secreting cytokines, chemokines, growth factors, and reactive oxygen species (1).

Although lymphocytes infiltrate into the lung following intratracheal bleomycin challenge, their role in the induction and perpetuation of the fibrotic response remains controversial. The administration of bleomycin into mice with severe combined immunodeficiency caused the development of pulmonary fibrosis, suggesting that lymphocytes are not required (7). In contrast, T cell depletion, T cell blocking, and nude mutation have markedly attenuated pulmonary fibrosis after bleomycin administration, suggesting a role of T cells (8, 9). Furthermore, μMT mice are resistant to silica-induced lung fibrosis, indicating a role of B cells in the development of pulmonary fibrosis, although B cells also had protective effects (10). Thus, the involvement of lymphocyte infiltration in the pathogenesis of pulmonary fibrosis remains unknown.

Recent studies of B cells have demonstrated that B cells regulate immunity via various ways that had not been previously appreciated. In addition to secreting immunoglobulins, B cells play roles such as antigen presentation, cytokine production, and the regulation of lymphoid organogenesis, effector T cell differentiation, and dendritic cell function (11, 12). Accordingly, critical roles of B cells have been demonstrated in several immune-mediated diseases. Because B cell fate and function are tightly regulated by signal transduction through the B cell receptor and functionally interrelated cell surface receptors, such as CD19, CD21, CD22, CD40, BAFF receptor, and Fcγ receptor IIb, modulation of these receptors can be a potential strategy for regulating these disorders (13, 14). Among them, CD19 is generally considered as a positive B cell response regulator (15). B cells from CD19-deficient (CD19−/−) mice have a significantly decreased capacity to proliferate in response to B cell mitogens, although B cells from human CD19 (hCD19)–transgenic mice have enhanced proliferative responses. Serum immunoglobulin levels are decreased in CD19−/− mice but are elevated in hCD19-transgenic mice. In addition, CD19−/− mice exhibit an attenuated humoral immune response, which is augmented in hCD19-transgenic mice. These reciprocal observations in mouse models have indicated that CD19 is a positive regulator of B cells and B cell–induced immune diseases (15). Because the roles of CD19 and B cells in pulmonary fibrosis are still unclear, their roles in experimental lung fibrosis were addressed in the current study.

MATERIALS AND METHODS

Antibodies and immunofluorescence analysis.

B220, CD19, CD21, and CD23 monoclonal antibodies (mAb) were purchased from BD PharMingen (San Diego, CA). CXCR3 mAb were purchased from R&D Systems (Minneapolis, MN). Single-cell suspensions of spleen were generated by gentle dissection. Viable cells were counted using a hemocytometer, with relative lymphocyte percentages determined by flow cytometric analysis. Blood erythrocytes were lysed after immunofluorescence staining using FACS Lysing Solution (BD Biosciences, San Jose, CA). Single-cell leukocyte suspensions were stained on ice using predetermined optimal concentrations of each antibody for 20 minutes, and fixed. Cells with the light scatter properties of lymphocytes were analyzed by 2–3-color immunofluorescence staining, with a FACScan or FACScanto flow cytometer (Becton Dickinson, San Jose, CA). Background staining was determined using nonreactive control mAb (Caltag, South San Francisco, CA), with gates positioned to exclude ≥98% of the cells.

Mice.

CD19−/− mice, hCD19-transgenic mice, and L-selectin−/− mice were produced as previously described (15, 16). Intercellular adhesion molecule 1–deficient (ICAM-1−/−) mice (17) expressing residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs including the lung (18), E-selectin−/− mice, and P-selectin−/− mice were from The Jackson Laboratory (Bar Harbor, ME). Mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1−/−) were generated as described previously (19). All mice were backcrossed between 5–10 generations onto the C57BL/6 genetic background. The mice used for the experiments were 12–16 weeks old. Age-matched C57BL/6 mice (The Jackson Laboratory) were used as controls with equivalent results; therefore, all control results were pooled. The size of the body or lungs was similar for mutant mice and their wild-type (WT) littermates. All mice were healthy, fertile, and did not display evidence of infection or disease. All mice were housed in a specific pathogen–free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science, and the Committee on Animal Experimentation of Nagasaki University Graduate School of Biomedical Sciences.

Bleomycin administration.

Bleomycin sulfate (Nippon Kayaku, Tokyo, Japan) was administered to mice that had been anesthetized by inhalation of diethyl ether. Using aseptic techniques, a single incision was made at the neck, and the muscle covering the trachea was snipped to expose the tracheal rings. A single intratracheal instillation of bleomycin sulfate (8 mg/kg [7.68 units/kg]) in 250 μl of sterile saline was performed using a 27-gauge needle.

Histologic examination and immunohistochemical analysis.

The same mice were used for histologic evaluation of fibrosis, measurement of hydroxyproline content, and counting the number of splenic B cells, while separate mice were used for the analysis of bronchoalveolar lavage fluid (BALF) components. After the right lung of each mouse was removed for hydroxyproline assay, the left lung was inflated and fixed with 3.5% paraformaldehyde and embedded in paraffin. Six-micrometer sections were stained with hematoxylin and eosin (H&E) to evaluate alveolitis and with Azan-Mallory stain to identify collagen deposition in the lung. Five H&E-stained sections of the entire lung from each mouse were chosen randomly. The severity of lung inflammation was determined using a semiquantitative scoring system as previously described (20). The pathology scores were defined as follows: 0 = no lung abnormality, 1 = presence of inflammation and fibrosis involving <25% of the lung, 2 = lesions involving 25–50% of the lung, and 3 = lesions involving >50% of the lung. The mean of the pathology scores for at least 5 sections was determined for individual mice.

Hydroxyproline assay.

Hydroxyproline is a modified amino acid uniquely found at a high percentage in collagen. Therefore, the tissue hydroxyproline content of lungs was assessed as a quantitative measure of collagen deposition, as previously described (8). The lung vasculature was perfused free of blood by slowly injecting 3 ml of phosphate buffered saline (PBS) into the right ventricle. The right lung was then excised and homogenized in 2 ml of PBS, pH 7.4, with a Tissue Tearor (Iuchi, Osaka, Japan). Each sample (0.5 ml) was desiccated overnight at 110°C and then digested in 1 ml of 6N HCl for 8 hours at 120°C. Samples were again desiccated for 6 hours at 120°C. Fifty microliters of citrate–acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% NaOH, 1.2% glacial acetic acid, pH 6.0) and 1 ml of chloramine T solution (1.13 gm chloramine T, 8 ml 1-propanol, 8 ml H2O, 64 ml citrate–acetate buffer) were added to each sample, and the samples were left at room temperature for 20 minutes. Next, 1 ml of Ehrlich's solution (10.13 gm p-dimethylaminobenzaldehyde, 41.85 ml 1-propanol, 17.55 ml 70% perchloric acid) was added and incubated for 15 minutes at 65°C. Samples were cooled for 10 minutes, spun at 3,100g for 5 minutes, and read at 550 nm on a spectrophotometer. A hydroxyproline standard solution of 0–4 mg/ml was used to generate a standard curve. Eight to ten mice of each genotype were examined. All reagents were purchased from Wako (Osaka, Japan).

Preparation of BALF.

BALF cells were prepared as described elsewhere (21). Briefly, at 2 and 7 days postinstillation, the mice were killed and the lungs lavaged with saline prior to fixation. BALF was collected as follows: 1 ml of saline was instilled 3 times and withdrawn from the lungs via an intratracheal cannula. In each mouse examined, ∼2.5 ml of BALF was retrieved. A 500-μl aliquot of the recovered BALF was analyzed for total and differential leukocyte counts. A total leukocyte count was performed using a hemocytometer in the presence of trypan blue.

Enzyme-linked immunosorbent assay (ELISA).

ELISA kits for interleukin-10 (IL-10), interferon-γ (IFNγ), IL-6 (MedSystems Diagnostics, Vienna, Austria), and CXCL10/IFNγ-inducible 10-kd protein, CXCL9/monokine induced by IFNγ, and CCL17/thymus and activation–regulated chemokine (R&D Systems), and immunoglobulin (Southern Biotechnology, Birmingham, AL) were used according to the manufacturers' protocols.

B cell stimulation.

Splenic B cells were purified by removing T cells with anti-Thy1.2 antibody–coated magnetic beads (Dynal, Lake Success, NY). Purified B cells were cultured in 0.2 ml of culture medium in 96-well flat-bottomed plates with lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO) or anti-CD40 mAb for 72 hours. CXCR3 expression was assessed by immunofluorescence analysis.

Statistical analysis.

The Mann-Whitney U test was used for determining the level of significance of differences in sample means, and the Bonferroni test was used for multiple comparisons.

RESULTS

Decreased pulmonary fibrosis in CD19−/− mice and augmented pulmonary fibrosis in hCD19-transgenic mice.

We estimated the role of CD19 in lung fibrosis using CD19−/− mice and hCD19-transgenic mice, which expressed human CD19 on B cells in addition to normal mouse CD19 (22). Because human CD19 can restore mouse CD19 function when expressed at physiologically appropriate cell surface sites, hCD19-transgenic mice have served as a mouse model for functional overexpression of CD19 (23). Sixteen days after bleomycin challenge, WT mice exhibited consolidation that consisted of subpleural foci of collapsed alveolar walls with dense inflammatory cell infiltration, decreased total lung capacity, and increased collagen deposition (Figures 1A and B). These pathologic changes were significantly reduced in CD19−/− mice (37% decrease; P < 0.005) (Figure 1C). Because none of hCD19-transgenic mice that received bleomycin still survived on day 11 (Figure 1E), we compared pathologic changes in 10 separate hCD19-transgenic mice at day 10 with such changes in WT mice at day 16.

Figure 1.

A and B, Representative histologic sections of lungs from CD19−/− mice and wild-type mice at 16 days and human CD19–transgenic (hCD19TG) mice 10 days following intratracheal injection of bleomycin (BLM). Mice that received an intratracheal injection of saline served as controls. The lung sections were stained with hematoxylin and eosin to evaluate alveolitis (A) and with Azan-Mallory stain to identify collagen deposition (blue color) (B). Results are representative of those obtained from 8–10 mice of each genotype. (Original magnification × 80.) C and D, Quantitative assessment of pulmonary fibrosis, as measured by the pathology score (C) and hydroxyproline content (μg/lung) (D) (day 16 for CD19−/− and wild-type mice, and day 10 for hCD19-transgenic mice). E, Time course of survival after intratracheal administration of BLM (n = 15 mice/group). Values in C are the mean and SD. Horizontal bars in D represent the mean. Values in E are the survival rate (%). ∗ = P < 0.05; ∗∗ = P < 0.005.

The pathologic changes were significantly enhanced by CD19 overexpression, even at earlier time points (Figures 1A and B). The pathology score was significantly increased in hCD19-transgenic mice in comparison with WT mice (136% increase; P < 0.05) (Figure 1C). However, lung sections from saline-treated mice from these 3 strains showed no significant change. Pulmonary fibrosis was further assessed by quantitatively measuring the hydroxyproline content in the lungs (Figure 1D). Sixteen days following bleomycin administration, the hydroxyproline content of lungs from WT mice was increased by almost 2.5-fold (P < 0.005) compared with that of lungs from saline-treated WT control mice (Figure 1D). The hydroxyproline content was significantly reduced in bleomycin-treated CD19−/− mice at day 16 (54% decrease; P < 0.005) and significantly increased in hCD19-transgenic mice 10 days after bleomycin administration (122% increase; P < 0.05) compared with bleomycin-treated WT mice (day 16). The hydroxyproline content of lungs was similar in saline-treated CD19−/− mice, WT mice, and hCD19-transgenic mice. Furthermore, CD19−/− mice showed a lower mortality relative to WT mice, whereas hCD19-transgenic mice showed a higher mortality compared with WT mice (Figure 1E). Thus, CD19 expression levels correlated with the severity of pulmonary fibrosis after intratracheal administration of bleomycin.

Effect of CD19 mutation on BALF B cell numbers.

Subsequent to acute alveolitis and interstitial inflammation, fibrotic responses occurred during the second week (24). Therefore, the number of inflammatory cells was assessed 2 days and 7 days after bleomycin challenge (Figure 2). The total number of leukocytes in BALF reached a maximum 7 days after bleomycin challenge in WT mice (24). The leukocyte influx on day 7 was significantly reduced in bleomycin-treated CD19−/− mice (72% decrease; P < 0.05) compared with bleomycin-treated WT mice (Figure 2A). However, the total number of leukocytes in CD19−/− mice remained significantly increased after 7 days of challenge in comparison with the number in saline-treated mice.

Figure 2.

A, Total cell number in 2.5 ml of bronchoalveolar lavage fluid (BALF). B and C, Numbers of B220+ B cells in BALF (B) and the spleen (C) from CD19−/−, wild-type, and human CD19–transgenic (hCD19TG) mice after bleomycin (BLM) challenge or saline administration (day 16 for wild-type and CD19−/− mice, and day 10 for hCD19-transgenic mice). Values are the mean and SEM results from 5 mice in each group. D, Expression of CD21 and CD23 by B220+ cells in BALF and spleen from wild-type and CD19−/− mice. ∗ = P < 0.05; ∗∗ = P < 0.001, versus wild-type.

To further assess the association of CD19 expression levels and B cell numbers in BALF, we compared the numbers of B220+ B cells on day 7 in BALF from CD19−/− mice, WT mice, and hCD19-transgenic mice. The number of B220+ B cells in BALF was significantly decreased in CD19−/− mice (82% decrease; P < 0.001) and significantly increased in hCD19-transgenic mice (180% increase; P < 0.001) compared with WT mice (Figure 2B). Nonetheless, the number of B220+ B cell in the spleen did not correlate with CD19 expression levels (Figure 2C). Collectively, B220+ B cell numbers in BALF were associated with CD19 expression levels. In addition, the expression of CD21 and CD23 by BALF B220+ B cells was similar to that of follicular B cells (a relatively mature population) in the spleen (Figure 2D). Thus, CD19 deficiency inhibited B cells from accumulating in the alveolar compartment after bleomycin challenge.

CD19-regulated B cell migration into the lung.

The number of BALF B220+ B cells after intratracheal bleomycin challenge is likely associated with B cell migration from the circulation, because only a small number of B cells existed in BALF before bleomycin challenge and shortly after the challenge (Figure 2B and data not shown). Nonetheless, there are few studies regarding B cell migration into sites of inflammation. Therefore, we addressed the relative contribution of adhesion molecules, which regulate migration of T cells as well as other leukocyte components into sites of inflammation, during bleomycin-induced B cell migration. The ratio of the number of B220+ B cells to the total cell number in BALF collected 7 days after bleomycin challenge was compared in adhesion molecule mutants (Figure 3A). There was no difference in the B220+ B cell number:total cell number ratio in BALF between adhesion molecule–deficient strains, including ICAM-1−/−, L-selectin−/−, ICAM-1/L-selectin−/−, E-selectin−/−, and P-selectin−/−, and WT mice (Figure 3A). In contrast, CD19 deficiency significantly reduced the ratio compared with that in WT mice, while CD19 overexpression significantly enhanced the ratio. In addition, mouse CD19 expression on circulating B220+ B cells was similar between ICAM-1−/−, L-selectin−/−, ICAM-1/L-selectin−/−, E-selectin−/−, P-selectin−/−, hCD19-transgenic, and WT mice (Figures 3B and C). Thus, functional CD19 expression levels correlated with B220+ B cell numbers in BALF.

Figure 3.

A, Ratio of the total number of B cells to the total cell number in BALF from CD19−/− mice, hCD19-transgenic mice, adhesion molecule–deficient mice, and wild-type mice. BALF was collected 7 days after intratracheal injection of bleomycin. Values are the mean and SEM results from at least 4 mice in each group. ∗ = P < 0.005 versus wild-type. B, CD19 expression on circulating B cells as assessed by indirect immunofluorescence staining and flow cytometric analysis. The fluorescence intensities of B220+ cells (solid histograms) or B220− cells (shaded histograms) stained with anti-CD19 mAb are shown on a 4-decade log scale. C, CD19 expression levels in circulating B220+ B cells as assessed by indirect immunofluorescence staining and flow cytometric analysis. ICAM-1 = intercellular adhesion molecule 1; mCD19 = mouse CD19; hCD19 = human CD19 (see Figure 2 for other definitions).

Relationship of B cell migration into the lung and CD19-induced CXCR3 up-regulation.

Next, we addressed CXCR3 expression on B cells in the circulation and BALF, 7 days after bleomycin or saline challenge (Figures 4A and B), because it has been suggested that migration of mature B cells and their malignant counterparts into sites of inflammation is dependent on CXCR3 (25–27). WT mice that had been challenged with bleomycin showed up-regulated CXCR3 expression on circulating B220+ B cells, while CD19−/− mice did not show this up-regulation (Figure 4A). The majority of B220+ B cells in BALF expressed CXCR3 (Figure 4B). Thus, CD19 up-regulated CXCR3 expression on B cells that had infiltrated into sites of inflammation.

Figure 4.

A and B, Representative fluorescence intensities of B220+ B cells stained with anti-CXCR3 mAb in blood or BALF 7 days after challenge with BLM or saline. Values are the mean and SEM results from at least 5 mice per group. C, CXCR3 up-regulation on B cells in response to anti-CD40 antibody or lipopolysaccharide (LPS). Purified B220+ splenocytes were cultured for 72 hours with the indicated amounts of anti-CD40 antibody or LPS. Values are the mean ± SEM results from 4 independent experiments. D, CXCL10, CXCL9, and CCL17 levels in BALF 7 days after BLM challenge or saline administration, as determined by enzyme-linked immunosorbent assay. Values are the mean and SEM results from 3 separate measurements of each sample and from at least 5 mice in each group. ∗ = P < 0.05; ∗∗ = P < 0.005, versus wild-type. MFI = mean fluorescence intensity; IP10 = interferon-γ–inducible 10-kd protein; MIG = monokine induced by interferon-γ; TARC = thymus and activation–regulated chemokine (see Figure 2 for other definitions).

To address whether this up-regulation of CXCR3 was related to CD19 expression, purified B cells from the spleens of CD19−/−, WT, and hCD19-transgenic mice were stimulated with anti-CD40 antibodies or LPS for 3 days. Stimulation with anti-CD40 antibodies or LPS up-regulated CXCR3 expression in WT mice (Figure 4C). The up-regulation of CXCR3 expression was augmented in hCD19-transgenic mice, while it was reduced in CD19−/− mice. Thus, CD19 signaling accelerated up-regulation of CXCR3 in response to B cell stimulation. Nonetheless, B cell migration was independent of the level of chemokines (including CXCL10, CXCL9, and CCL17), because these levels in BALF after bleomycin challenge were similar in CD19−/−, WT, and hCD19-transgenic mice (Figure 4D). Thus, B cell migration into the lung correlated in part with the CXCR3 up-regulation that was accelerated by CD19 signal transduction.

Correlation between IL-6 release in BALF after bleomycin administration and CD19 expression levels.

We next addressed the cytokine levels in BALF 7 days after bleomycin challenge. IL-6 and IL-10 have been shown to be possible soluble mediators of fibrosis in patients with systemic sclerosis, in whom overexpression of CD19 levels may be associated with fibrosis in skin and the lung (28). IFNγ is a protective mediator of human pulmonary fibrosis (29). In the absence of bleomycin challenge, BALF contained only negligible quantities of IL-6, IL-10, and IFNγ (Figure 5 and data not shown). However, BALF from bleomycin-treated WT mice exhibited increased amounts of IL-6, while IL-6 levels were significantly reduced in bleomycin-treated CD19−/− mice (P < 0.005) (Figure 5A). IL-6 levels were significantly elevated in bleomycin-treated hCD19-transgenic mice compared with the levels in bleomycin-treated WT mice (P < 0.05) (Figure 5A). However, IL-6 levels following bleomycin challenge were similar in ICAM-1−/− mice, L-selectin−/− mice, L-selectin/ICAM-1−/− mice, E-selectin−/− mice, P-selectin−/− mice, and WT mice (Figure 5C). Thus, IL-6 production after bleomycin administration correlated with CD19 expression levels. BALF IL-10 levels were significantly increased in bleomycin-treated WT mice compared WT mice that did not receive bleomycin. IL-10 levels in bleomycin-treated CD19−/− mice and WT mice were similar, while IL-10 levels were significantly reduced in bleomycin-treated hCD19-transgenic mice compared with the levels in bleomycin-treated WT mice (P < 0.05). In addition, BALF IFNγ levels were not detectable in WT mice, CD19−/− mice, and hCD19-transgenic mice, even after administration of bleomycin (data not shown).

Figure 5.

Interleukin-6 (IL-6) and IL-10 levels in bronchoalveolar lavage fluid 7 days after bleomycin (BLM) challenge or saline administration. IL-6 and IL-10 levels were measured by enzyme-linked immunosorbent assay. Values are the mean and SEM results from 3 separate measurements of each sample and from at least 5 mice in each group. ∗ = P < 0.05 versus BLM-treated wild-type mice; ∗∗ = P < 0.005. hCD19TG = human CD19–transgenic; ICAM-1 = intercellular adhesion molecule 1.

Correlation between CD19 expression and immunoglobulin levels in BALF after bleomycin administration.

The effects of the CD19 mutation on B cell responses during bleomycin-induced pulmonary fibrosis were assessed by determining the immunoglobulin levels in BALF. Human CD19–transgenic mice had significantly elevated levels of IgM (P < 0.005), IgG2b (P < 0.005), and IgG2c (P < 0.003) 7 days after bleomycin challenge compared with those in WT mice, while IgG1 and IgG3 levels in hCD19-transgenic mice were not different from those in WT mice (Figure 6). In contrast, CD19−/− mice with bleomycin challenge had significantly decreased levels of IgM (P < 0.05), IgG1 (P < 0.003), and IgG3 (P < 0.05) compared with WT mice with bleomycin administration. Thus, IgG levels in BALF after bleomycin challenge correlated with CD19 expression levels.

Figure 6.

Immunoglobulin levels in bronchoalveolar lavage fluid from CD19−/−, wild-type, and human CD19–transgenic (hCD19TG) mice, 7 days after bleomycin (BLM) challenge or saline administration, as determined by isotype-specific enzyme-linked immunosorbent assay. Horizontal bars represent the means.

DISCUSSION

The results of the present study suggest that CD19 plays a crucial role in the development of bleomycin-induced pulmonary fibrosis. CD19 deficiency significantly inhibited bleomycin-induced pulmonary fibrosis (Figure 1), while CD19 overexpression augmented pulmonary fibrosis. Furthermore, CD19 expression levels positively correlated with the accumulation of B cells in BALF (Figure 2). Because the number of B cells was very small in saline-treated mice, B cells from the circulation are likely to accumulate into the lung space after bleomycin administration (30). In addition, the levels of IL-6 and immunoglobulin in BALF correlated with CD19 expression levels and the severity of pulmonary fibrosis. These results suggest that CD19 regulates the development of bleomycin-induced pulmonary fibrosis by modulating B cell functions, including migration into the sites of inflammation, cytokine release, and immunoglobulin production. However, the process beyond the day 16 time point and administration of various doses of bleomycin will provide further information concerning the association between CD19 function and pulmonary fibrosis.

In the current system using mice with genetically modified CD19 expression, the expression of CD19 was demonstrated to be associated with a predisposition for lung injury and pulmonary fibrosis. However, in addition to B cells, a subset of dendritic cells was shown to express CD19 and to have a possible regulatory role in pulmonary fibrosis (31, 32). Although the function of CD19-expressing dendritic cells remains unknown in the current study, a lack of CD19 on these cells did not seem to influence the development of pulmonary fibrosis, because no functional defect has been observed in T cells and dendritic cells from CD19−/− mice (33). In addition, the levels of B cell activation markers, including IL-6, and immunoglobulin were associated with the severity of pulmonary fibrosis and CD19 expression levels (Figures 5 and 6). Therefore, it is suggested that the predisposition for pulmonary fibrosis is preferentially associated with B cell CD19 expression. Recent studies have demonstrated that B cells regulate immune responses in various ways in addition to immunoglobulin secretion. Therefore, infiltrated B cells could play a role in the development of fibrosis by humoral immunity–dependent and independent pathways. Consistent with this possibility, long-term B cell activation resulting from augmented CD19 signaling in tight skin mice, a genetic model for systemic sclerosis, has led to fibrosis through overproduction of IL-6 (34).

Overexpression of CD19 has been associated with fibrosis in systemic sclerosis (28). B cell infiltration into the lung is associated with pulmonary fibrosis in patients with systemic sclerosis (35). In the present study, infiltrated B cells may produce IL-6 and immunoglobulin, which may induce pulmonary fibrosis, because IL-6 levels and immunoglobulin levels in BALF correlated with the severity of pulmonary fibrosis (Figures 1C and 5). In a previous study, CD19 hyperexpression augmented Sle1-induced autoantibody production but did not enhance clinical nephritis in a lupus model (30). However, a small increase in CD19 expression (∼20%) is related to fibrosis in patients with systemic sclerosis (28, 36). Thus, it is still unclear whether CD19 expression affects only autoantibody production or whether it induces a disease phenotype in autoimmune disorders. Although the current study is limited in that it was unable to elucidate the mechanisms of how B cells affect disease phenotype, the current results suggest a role of CD19 in the development of pulmonary fibrosis. In this regard, it will be worthwhile to investigate the role of B cell activation in pulmonary fibrosis, focusing on the autoantibody profile, isotype, B cell–related cytokines, and other B cell–associated functions.

The interstitial lung diseases include a diverse set of disorders in which pulmonary inflammation and fibrosis are the final common pathologic manifestations. Thus, fibrotic change is considered, if not exclusively, as the end result of inflammatory reactions induced by a variety of stimuli (37). In general, leukocyte recruitment into sites of inflammation is achieved using constitutive or inducible expression of multiple molecules, including L-selectin, P-selectin, E-selectin, ICAM-1, vascular cell adhesion molecule 1, very late activation antigen 4, CD44, hyaluronic acid, and lymphocyte function–associated antigen 1, and chemokine receptors (38–41). However, very few studies have investigated which molecules are involved in B cell migration into sites of inflammation (25, 26). Antigen-specific IgG-secreting cells in the spleen temporarily up-regulate their expression of CXCR3 and responsiveness to the CXCR3 ligands expressed on endothelial cells at the site of inflammation (42). Thus, circulating B cells that express CXCR3 can directly enter the sites of inflammation from the blood. In the current study, up-regulated CXCR3 expression on circulating B cells was observed in WT mice that underwent bleomycin challenge, while such up-regulation was not observed in CD19−/− mice (Figure 4A). In addition, a majority of BALF B cells expressed CXCR3 (Figure 4B). Furthermore, CD19 levels correlated with CXCR3 up-regulation in response to B cell stimulation (Figure 4C). Therefore, it is possible that CXCR3 was up-regulated by bleomycin-induced B cell stimulation, which interacts with CD19 signal transduction. As a result, B cells can migrate into sites of inflammation.

Collectively, the current results suggest that CD19 and/or its downstream effecter molecules regulate B cell migration, although further studies, including adoptive transfer experiments in combination with CXCR3−/− mice and CXCR3-blocking antibodies, are needed. In this regard, the present model system could provide an original approach to elucidate the contribution of adhesion molecules during B cell–mediated inflammation.

Recently, technologic advances and molecular elucidation of immune systems have introduced mAb into the therapy for autoimmune diseases. Furthermore, an increasing number of reports have suggested the role of B cells in the pathophysiology of autoimmune diseases, including rheumatoid arthritis, lupus, and systemic sclerosis (11). Although several mAb targeting B cell function are ready for use in several autoimmune disorders (43–49), the results of the current study should not be translated to a straightforward suggestion that B cell– or CD19-targeting therapy is a new optional approach to pulmonary fibrosis. Because several subsets and various regulators of B cells have been introduced (15, 50), multifaceted knowledge and careful designing will be needed for security and efficiency.

AUTHOR CONTRIBUTIONS

Dr. Tedder had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Komura, Sato.

Acquisition of data. Komura, Yanaba, Horikawa.

Analysis and interpretation of data. Komura, Ogawa, Fujimoto, Tedder, Sato.

Manuscript preparation. Komura, Sato.

Statistical analysis. Komura, Sato.

Acknowledgements

We thank Ms M. Matsubara, Y. Yamada, A. Usui, M. Yozaki, and K. Shimoda for technical assistance.

Ancillary