Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Superantigens are extremely potent polyclonal mitogens that stimulate a large proportion of T cells expressing specific T-cell receptor (TCR) Vs in both mice and humans (1, 2), and they have been recently suggested to participate in the development of various autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes mellitus (IDDM), and psoriasis (3).

The pathogenesis and mechanisms underlying the development of interstitial pneumonia associated with autoimmune disease are not yet known (6, 7). We have previously reported that an intratracheal instillation of superantigen induced interstitial pneumonia in autoimmune-prone mice but not in non-autoimmune-prone mice (8). Because bronchoalveolar lavage fluid (BALF) can be used to demonstrate the pathophysiology of interstitial pneumonia to some degree (9, 10), we analyzed the BALF in our murine model of interstitial pneumonia. We found that the instilled superantigen, staphylococcal enterotoxin B (SEB)- reactive T cells, neutrophils, and alveolar macrophages significantly increased in BALF from MRL +/+ mice injected with SEB 3 d previously (Day 3). A reverse transcriptase/polymerase chain reaction (RT-PCR) analysis in BALF from MRL +/+ mice revealed early increases in messenger RNA (mRNA) of several chemokines and cytokines after the SEB administration. These results suggest that the instilled superantigen directly stimulated the resident cells in bronchoalveolar space and recruited inflammatory cells to the bronchoalveolar space and lung parenchyma around such spaces.

We also examined the effects of corticosteroid and FK506, immunosuppressive agents, to investigate immunologic events in the development of interstitial pneumonia in this animal model. Corticosteroid is a powerful anti-inflammatory agent that alters the transcription of many genes (11, 12). FK506 is a macrolide compound isolated from Streptomyces tsukubanesis and has potent immunosuppressive properties (13, 14). In our present experiment, FK506 but not corticosteroid significantly suppressed SEB-induced T-cell expansions and resulted in the prevention of interstitial pneumonia. Further, interstitial pneumonia developed in SCID mice administered T cells from MRL +/+ mice and SEB. These results indicate that superantigen-reactive T cells play a critical role in the initiation of interstitial pneumonia.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Antibodies and Reagents

Fluorescein isothiocyanate (FITC)-conjugated antimouse CD4 monoclonal antibody (mAb), FITC-conjugated antimouse CD8 mAb, biotin-conjugated antimouse CD8 mAb, biotin-conjugated antimouse V6 TCR mAb, and biotin-conjugated antimouse V8 TCR mAb were purchased from PharMingen Co. (San Diego, CA). Antimouse macrophage mAb was purchased from BMA Biomedicals Ltd. (Augst, Switzerland). Streptavidin/R-phycoerythrin (Ancell Co., Bayport, MN) was used at a dilution of 1:25. Magnetic beads coated with antimouse CD4 mAb, antimouse CD8 mAb, or antirat immunoglobulin (Ig)G were purchased from Dynal A. S. (Oslo, Norway). SEB (Sigma Chemical Co., St. Louis, MO) was dissolved in phosphate-buffered saline (PBS) at 1 mg/ml. FK506 was generously supplied by Fujisawa Pharmaceutical Co. (Osaka, Japan). FK506 and prednisolone sodium succinate (Shionogi Co., Osaka, Japan) were dissolved in saline.

Mice

Female MRL +/+ (H-2^k), 12 wk of age, were purchased from SLC Japan, Inc. (Shizuoka, Japan). Female C.B-17 SCID mice, 6 to 8 wk of age, were purchased from CLEA Japan, Inc. (Tokyo, Japan). All mice were maintained in a specific pathogen-free environment at the Laboratory Animal Center of the Kumamoto University School of Medicine. SCID mice were housed in sterilized microbarrier units under germ-free conditions.

Administration of SEB

The administration of SEB into the tracheas of the MRL +/+ mice was performed as previously described (8). In brief, each mouse was anesthetized with an intraperitoneal injection of 0.34 to 0.42 ml (60 µg/g body weight) of sodium pentobarbital (Abbott Laboratories, North Chicago, IL). After the trachea was exposed, a 27-gauge needle was inserted into the trachea and 40 µg of SEB or 40 µl of PBS was instilled on Day 0.

BALF

BALF was obtained from the lungs of mice that had received the intratracheal instillations of SEB 1, 3, 7, or 14 d previously. The mice were deeply anesthetized and killed by exsanguination of the abdominal aorta. After the chest was opened, a 20-gauge plastic catheter was inserted into the trachea and tied with 5-0 silk. The lungs from each animal were lavaged through a catheter with 1 ml of chilled PBS three times. The cells were pelleted by centrifugation and counted using a hemacytometer. Differential counts were performed on Diff-Quik-stained preparation. The rest of the pellets were used for the flow cytometric analysis or for the extraction of mRNA.

Flow Cytometry

Peripheral blood mononuclear cells (PBMCs) from SEB- or PBS-treated mice were purified by gradient centrifugation using Lympholyte-M (Cedarlane, ON, Canada) and spleen cells were removed and treated with Tris-buffered 0.16 M ammonium chloride to lyse erythrocytes. Cells in BALF, spleen, lymph node, and PBMCs were stained with FITC-conjugated antimouse CD4 mAb or FITC-conjugated antimouse CD8 mAb together with biotinylated antimouse V6 TCR mAb or antimouse V8 TCR mAb, followed by incubation with streptavidin/R-phycoerythrin. The cell analysis was performed on a FACScan flow cytometer (Becton Dickinson Co., San Francisco, CA).

RT-PCR

The extraction of mRNA was performed from the isolated macrophages, CD4⁺ T cells, and CD8⁺ T cells in BALF, as well as the whole cells in BALF. To separate macrophages, CD4⁺ and CD8⁺ T cells and BALF cells were incubated with the magnetic beads coated with anti-CD4 mAb or anti-CD8 mAb, or were incubated with rat antimouse macrophage mAb followed by the addition of magnetic beads coated with antirat IgG Ab, and isolated with a magnetic field generated by a magnetic particle concentrator (Dynal A. S.). mRNA was directly extracted from the cells of BALF using a Micro-Fasttrack mRNA isolation kit (Invitrogen, San Diego, CA). A total of 1 µg of mRNA was reverse transcribed into complementary DNA (cDNA) using an oligo(dT)_12-18 primer (GIBCO BRL, Rockville, MD) and moloney murine leukemia virus RT (GIBCO BRL) in a 20-µl reaction, followed by PCR amplification. Each reaction contained 1 µl of cDNA, 500 nM of each primer (Table 1), 200 mM of each of the four deoxynucleotide triphosphates, 1.5 mM MgCl₂, and 1.25 U of DNA polymerase, AmpliTaq (Perkin-Elmer, Foster City, CA) in a final volume of 50 µl reaction mixture. After a 2-min initial denaturing at 94°C, 33 or 35 cycles of denaturation (94°C) for 60 s, annealing (55°C) for 60 s, and extension (72°C) for 90 s were conducted. As an internal control, -actin was amplified to normalize the starting amount of cDNA for each sample. The optimal numbers of PCR cycles for exponential amplification were 33 for -actin and 35 for other cytokines and chemokines. Aliquots of the PCR reaction products (10 µl) were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining and ultraviolet transillumination. The resulting images were density-scanned (FAS-II; Toyobo, Osaka, Japan). Band intensities were determined by use of the NIH image 1.61 program (Wayne Rasband Analytics; National Institutes of Health, Bethesda, MD) and the cytokine or chemokine to -actin ratios were calculated.

Histology

Lung tissue was removed 7 d after intratracheal instillation of SEB, and fixed-inflated to 20 to 23 cm H₂O pressure with 10% formalin. After fixation, all lobes of the lung were sectioned by sagittal cuts. The tissue specimens were set in paraffin blocks, sectioned, and stained with hematoxylin and eosin (H&E) and azan. The slides of lung tissue were analyzed by a previously published method (8). Categories of histopathologic findings were as follows: (1) increased cellularity into the alveolar septal walls, (2) inflammatory cell accumulation in the periarterial space, and (3) increase in pulmonary interstitial collagen fibers. Semiquantitative scoring was as follows: 0 = no change; 1 = slight change; 2 = moderate change; 3 = marked change; and 4 = severe change.

Enzyme-Linked Immunosorbent Assay

Tumor necrosis factor (TNF)-, interferon (IFN)-, and interleukin (IL)-2 in BALF from SEB- or PBS-treated MRL +/+ mice were detected by enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Camarillo, CA), following the manufacturer's instructions. The sensitivity of each ELISA kit was as follows: TNF-, < 3.0 pg/ml; IFN-, < 1.0 pg/ml; and IL-2, < 13 pg/ml.

Administration of FK506 and Prednisolone

MRL +/+ mice were intraperitoneally injected with saline (saline group), FK506 (10 mg/kg/d; FK506 group), or prednisolone sodium succinate (2 mg/kg/d; prednisolone group) daily from the day before SEB administration to Day 3 or 7. The dosage regimen of FK506 was comparable to those used in previous studies (15, 16).

In Vitro Assay for Immunosuppressive Activity of FK506 and Prednisolone

Spleen cells from the nonstimulated MRL +/+ mice were treated with Tris-buffered 0.16 M ammonium chloride and cultured with 10 µg/ml SEB for 72 h in RPMI 1640 medium containing penicillin G (100 U/ml), streptomycin (100 µg/ml), 10% heat-inactivated fetal calf serum, and 2-mercaptoethanol (50 µm) in a 96-well culture plate at 10⁵ cells/100 µl (8, 17) in the presence of FK506 or prednisolone. A total of 10 µl of TetraColor One (5 mM tetrazolium monosodium sodium and 0.2 mM 1-methoxy-5-methylphenazinium methylsulfate; Seikagaku Corp., Tokyo, Japan) was added to each well and incubated for the final 4 h, and the plate was analyzed on an ELISA reader at 450 nm.

Cell Transfer into SCID Mice by Intratracheal Injection

Spleen T cells, T cell-depleted spleen cells, or BALF cells from untreated MRL +/+ mice were intratracheally injected into the lung of SCID mice with 40 µg of SEB. For T-cell purification, spleen cells treated with ammonium chloride were cultured on plastic dishes, then nonadherent cells were recovered and subjected to nylon wool columns (> 95% CD3⁺ T cells) (18). For T-cell removal, spleen cells were incubated with magnetic beads coated with anti-CD4 mAb and anti-CD8 mAb, then removed by magnetic extraction (< 2% CD3⁺ T cells). A total of 5 × 10⁵ cells of each group, suspended in 30 µl of minimum essential medium (MEM) was transferred into SCID mice. At 7 or 14 d after the cell transfer, SCID mice were killed and subjected to histopathologic examination.

Statistical Analysis

The values shown are means ± standard error of the mean (SEM). The statistical analysis was performed using the unpaired Student's t test. P values less than 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of Cells in BALF

As indicated in Figure 1, more than 98% of the cells in BALF from the PBS-treated MRL +/+ mice were macrophages. In BALF from the SEB-treated mice, the numbers of total cells, macrophages, lymphocytes, and neutrophils increased significantly, and peaked 3 d after the SEB administration. A slight increase in neutrophils was seen at Day 1 (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1.   Characterization of BALF cells from SEB- and PBS-treated mice. SEB administration increased the number of inflammatory cells in BALF. The numbers of total cells, macrophages, lymphocytes, and neutrophils in BALF obtained from SEB- and PBS-treated MRL +/+ mice at indicated days are shown. Data are means ± SEM. *P < 0.05 and **P < 0.005 versus control values obtained with PBS-treated mice at the same time point. (PBS-treated mice: Day 3, n = 5; Day 7, n = 5; Day 14, n = 5. SEB-treated mice: Day 3, n = 8; Day 7, n = 6; Day 14, n = 6.)

The fluorescence-activated cell sorter (FACS) analysis of BALF cells from the SEB-treated mice showed CD8⁺ T cells to be more abundant than CD4⁺ T cells, and the ratio of CD4/CD8 was 0.32 ± 0.12 at Day 3. An increase in SEB-reactive V8⁺ T cells was seen in both CD4⁺ and CD8⁺ T cells (Figure 2). Only a small number of V6⁺ T cells did not react to SEB.

View larger version (40K): [in this window] [in a new window]   Figure 2.   T-cell subpopulations in BALF from SEB-treated MRL +/+ mice. Cells in BALF obtained from MRL +/+ mice injected with SEB 3 d previously were stained with mAbs against CD4, CD8, V6 TCR, and V8 TCR as described in MATERIALS AND METHODS. Numbers indicate the percentage of cells in each quadrant. These data are representative of three individual experiments.

Phenotypic Analysis of Spleen T Cells

FACS analysis of surface markers on spleen T cells from PBS- and SEB-treated mice at Day 3 revealed that SEB treatment decreased the relative proportion of both CD4⁺ and CD8⁺ T cells expressing TCR V8 (Table 2). In PBMCs and lymph nodes, similar results were seen (data not shown).

Cytokine and Chemokine mRNA Expression of BALF Cells

We analyzed the cytokine and chemokine mRNA expressions in BALF cells by RT-PCR. The TNF- and IFN- mRNA levels in the SEB-treated mice were elevated at Day 3 compared with the PBS-treated mice, and then decreased at Day 14 (Figure 3A). The increase in mRNA expression of TNF- was observed mainly in macrophages and CD8⁺ T cells, and increased mRNA expression of IFN- was seen mainly in CD8⁺ T cells 3 d after SEB administration (Figure 3B). The expression of JE/monocyte chemoattractant protein (MCP)-1, regulated on activation, normal T cells expressed and secreted (RANTES), and transforming growth factor (TGF)- mRNA was detected at Day 3 and continued for 14 d in the SEB-treated mice. The peak of expression of platelet-derived growth factor (PDGF) mRNA was seen at Day 14 (Figure 3A). The expression of IL-4 and IL-5 mRNA did not increase at Day 3 or 14 (data not shown). In BALF 24 h after the SEB administration, slight increases in the mRNA expressions of TNF- and KC/gro were seen, but there was no increase in the mRNA of other cytokines or chemokines (data not shown).

View larger version (67K): [in this window] [in a new window]   View larger version (75K): [in this window] [in a new window]   Figure 3.   RT-PCR analysis of cytokine and chemokine mRNA in BALF cells. mRNA was extracted from whole BALF cells obtained from MRL +/+ mice treated with PBS or SEB 3 or 14 d previously, followed by RT-PCR for TNF-, IFN-, JE/MCP-1, RANTES, KC/gro, TGF-, PDGF, and -actin as an internal control (A). Alternatively, macrophages and CD4+ and CD8+ T cells were separated from BALF of MRL +/+ mice treated with SEB 3 d previously by magnetic beads coated with each Ab. mRNA from these cells was extracted and subjected to RT-PCR (B).

Effects of FK506 and Prednisolone In Vivo

To examine the effects of FK506 and prednisolone on SEB-induced interstitial pneumonia, mice were intraperitoneally injected with saline, FK506, or prednisolone daily. In the BALF from the FK506 group, the increases in the numbers of total cells, macrophages, lymphocytes, and neutrophils were inhibited at both Day 3 and Day 7 (Figure 4). The mRNA expressions of TNF-, IFN-, TGF-, RANTES, and JE/MCP-1 at Day 3 and those of RANTES, JE/MCP-1, and PDGF at Day 7 were significantly suppressed by FK506 treatment (Figure 5 and Table 3). The mean values of TNF-, IFN-, and IL-2 in BALF at Day 3 detected by ELISA were also suppressed by FK506 treatment (Figure 6). In BALF 24 h after SEB administration, there were no increases in the values of TNF-, IFN-, and IL-2 (data not shown). Histopathologically, FK506 significantly reduced both the cell infiltration into the alveolar septal walls and the synthesis of pulmonary collagen fibers; however, it did not reduce the inflammatory cell accumulation in the periarterial space (Figures 7 and 8). In contrast, prednisolone had no effect on the BALF or histologic findings (Figures 4, 7, and 8).

View larger version (37K): [in this window] [in a new window]   Figure 4.   The effects of FK506 and prednisolone on BALF cells. The numbers of total cells, macrophages, lymphocytes, and neutrophils in BALF from saline-, FK506-, and prednisolone-treated mice were examined 3 or 7 d after SEB administration. FK506 suppressed the inflammatory cell accumulation in the BALF of SEB-treated MRL +/+ mice. Data are means ± SEM. *P < 0.05 and **P < 0.005 versus control values obtained with the saline group at the same time point. (Saline group: Day 3, n = 6; Day 7, n = 6. FK group: Day 3, n = 6; Day 7, n = 5. Prednisolone group: Day 3, n = 6; Day 7, n = 6.)

View larger version (60K): [in this window] [in a new window]   Figure 5.   The effect of FK506 on the expression of cytokine and chemokine mRNA in BALF cells. mRNA was extracted from BALF cells obtained from the saline and FK506 groups. A semiquantitative RT-PCR analysis of TNF-, IFN-, JE/MCP-1, RANTES, KC/gro, TGF-, and PDGF expression in BALF cells from each was done. FK506 suppressed the expression of cytokine and chemokine mRNA in BALF cells from SEB-treated MRL +/+ mice. -actin primers were used as an internal control.

View larger version (15K): [in this window] [in a new window]   Figure 6.   The effect of FK506 on cytokine concentration in BALF from SEB-instilled MRL +/+ mice. FK506 suppressed IL-2, TNF-, and IFN- levels in the BALF from SEB-treated mice. The individual values and means of IL-2, TNF-, and IFN- in the BALF obtained from PBS-instilled MRL +/+ mice and the saline and FK506 groups at Day 3 are shown. *P < 0.05; **P < 0.01 versus the other group.

View larger version (92K): [in this window] [in a new window]   Figure 7.   Histopathologic findings in the lungs from saline-, FK506-, and prednisolone-treated mice. Lung tissue obtained from saline- (A, D), FK506- (B, E), and prednisolone- (C, F ) treated animals at Day 7 is shown. Lung tissue was stained with H&E (A, B, C) and azan (D, E, F ). Significant inflammatory cell infiltration and synthesis of collagen fibers in alveolar septal walls are seen in lung tissue from the saline and prednisolone groups. Accumulation of inflammatory cells around the arteries is seen in lung tissue from all groups. Bar = 50 µm.

View larger version (11K): [in this window] [in a new window]   Figure 8.   The effect of FK506 and prednisolone on histopathologic findings. Lung tissue obtained from the saline-, FK-, and prednisolone-treated mice at Day 7 was analyzed for the three categories and scoring system as described in MATERIALS AND METHODS. FK506 suppressed both cell infiltration into the alveolar septal walls (A) and the synthesis of pulmonary collagen fibers (C), but not the inflammatory cell accumulation in periarterial space (B). Each data point represents an individual mouse. * P < 0.05 versus the saline group.

Analysis of Immunosuppressive Activity of FK506 and Prednisolone In Vitro Assay

We compared the immunosuppressive effects of FK506 and prednisolone in vitro on SEB-induced spleen cell proliferation. FK506 inhibited the proliferation of spleen cells at the concentration of 0.1 ng/ml, whereas prednisolone required 100 ng/ml. In in vitro assay, prednisolone was approximately 1,000-fold less effective than FK506 in preventing SEB-induced spleen cell expansion (Figure 9).

View larger version (15K): [in this window] [in a new window]   Figure 9.   In vitro assay for immunosuppressive activity of FK506 and prednisolone. Spleen cells from nontreated MRL +/+ mice were cultured for 72 h with SEB (10 µg/ml) in the presence of FK506 or prednisolone. The suppressive effect of FK506 and prednisolone on SEB-induced spleen cell proliferation was analyzed with an ELISA reader at 450 nm after coincubation with TetraColor One. Data are means of triplicated samples ± SEM.

After 72 h stimulation of spleen cells with SEB (10 µg/ml), the CD4⁺/CD8⁺ ratio was 0.88 and the percentages of CD4⁺V8⁺/CD4⁺ T cells and CD8⁺V8⁺/CD8⁺ T cells were 39.4 and 51.7%, respectively, indicating the decrease in the CD4⁺/CD8⁺ ratio and the increase of proportions of V8⁺ T cells in both CD4⁺ and CD8⁺ T cells compared with freshly isolated spleen cells from nontreated and PBS-treated MRL +/+ mice (data not shown and Table 2).

Induction of Interstitial Pneumonia in SCID Mice

FK506 is known to act on other types of cells, such as macrophages and neutrophils, as well as T cells (19). Inasmuch as FK506 suppressed the induction of interstitial pneumonia, next we transferred T cells of MRL +/+ mice and SEB into SCID mice and examined whether the interstitial pneumonia developed to further investigate the role of T cells in this animal model. In SCID mice administered T cells from MRL +/+ mice and SEB, the cell accumulation into the perivascular space and the alveolar septal walls was seen. The synthesis of pulmonary collagen fibers was also detected, though the degree of it was weaker than that of SEB-treated MRL +/+ mice. In SCID mice injected with T cell-depleted spleen cells and SEB, or BALF cells and SEB, no histologic change was observed. Although slight accumulation of inflammatory cells into the perivascular space was seen in SCID mice injected with spleen T cells without SEB, there was a significant difference in the degree of histopathologic change between this group and the group injected with T cells and SEB (Figures 10 and 11).

View larger version (48K): [in this window] [in a new window]   Figure 10.   Histopathologic findings in the lungs from SCID mice transferred with spleen cells from MRL +/+ mice with or without SEB. Lung tissue obtained from SCID mice received spleen T cells of MRL +/+ mice and SEB (A), T cell-depleted spleen cells and SEB (B), and spleen T cells without SEB (C) at Day 14 is shown. Lung tissue was stained with H&E. Inflammatory cell infiltration and synthesis of collagen fibers in alveolar septal walls are seen in the SCID mice-transferred T cells and SEB (A), and slight cell accumulation of inflammatory cells in periarterial space is seen in the SCID mice-transferred T cells (C). Bar = 50 µm.

View larger version (17K): [in this window] [in a new window]   Figure 11.   Induction of interstitial pneumonia by the transfer of spleen T cells from MRL +/+ mice and SEB in SCID mice. Lung tissue obtained from SCID mice transferred with spleen T cells from MRL +/+ mice and SEB, T cell-depleted spleen cells and SEB, BALF cells and SEB, and spleen T cells without SEB at Day 14 was analyzed for the three categories and scoring system as described in MATERIALS AND METHODS. Inflammatory cell accumulation in the alveolar septal walls (A) and the periarterial space (B), but not the synthesis of collagen fibers (C), was significantly increased in the SCID mice that received T cells and SEB. Each data point represents an individual mouse. *P < 0.05; **P < 0.005 versus the other group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Superantigens are produced by many different pathogens, including bacteria, mycoplasma, and viruses (2, 20). Superantigens bind to both specific V segments of TCR and major histocompatibility complex molecules without being processed and stimulate very large numbers of T cells (1). The recently discovered selective expansion of T cells bearing specific TCR Vs in the synovial fluid of rheumatoid arthritis patients and islet of IDDM patients has suggested that superantigens participate in the development of various autoimmune diseases (3, 4).

In the present study, we analyzed the BALF in our murine model of interstitial pneumonia. Significant increases in the numbers of macrophages, lymphocytes, and neutrophils peaking 3 d after SEB treatment were observed. The analysis of the TCR V of infiltrated lymphocytes in BALF from SEB-treated MRL +/+ mice revealed expansions in SEB-reactive CD8⁺V8⁺ T cells. On the other hand, CD8⁺V8⁺ T cell number was decreased in spleen 3 d after SEB administration compared with PBS-treated mice. The stainings of spleen cells at Day 3 with anti-V8 mAb and Annexin V showed no significant increase in the proportion of apoptotic V8⁺ T cells (data not shown). These results suggest recruitment of CD8⁺V8⁺ T cells to lung tissue. Further, when the spleen cells were stimulated with SEB, CD8⁺V8⁺ T cells propagated much more than did CD4⁺V8⁺ T cells (data not shown). The accumulation of CD8⁺V8⁺ T cells in bronchoalveolar space after SEB administration may be due to proliferation of SEB-reactive T cells in bronchoalveolar space as well as to recruitment. The higher susceptibility of CD8⁺ T cells to SEB-induced T-cell proliferation than CD4⁺ T cells (data not shown) may contribute to the increase of CD8⁺ T cells in bronchoalveolar space. We also observed early increases in the expression of TNF-, IFN-, JE/MCP-1, RANTES, KC/gro, and TGF- mRNA in BALF cells. Further, mRNA expression of cytokines and chemokines was mainly increased in CD8⁺ T cells as well as in macrophages in BALF. Nagai and Izumi previously reported increases in CD8⁺ T cells in BALF from patients with autoimmune diseases (7). Although the role of accumulated CD8⁺ T cells in the bronchoalveolar space of this animal model has not been elucidated, we found that these CD8⁺ T cells produce proinflammatory cytokines and they may participate directly in the process of tissue destruction by several death systems, such as the CD95-CD95 ligand system. These results indicate that instilled SEB may first stimulate resident SEB-reactive T cells and some macrophages (21) in the bronchoalveolar space, and that these cells strongly proliferate and/or produce several proinflammatory cytokines and chemokines, resulting in the recruitment of several types of inflammatory cells to the lung.

TNF- mRNA was detected mainly in CD8⁺ T cells and macrophages at Day 3 in our model. TNF- is a cytokine with both inflammatory and fibrogenic activities (22). TNF- mRNA and protein have been detected in lungs from patients with idiopathic pulmonary fibrosis (IPF) (23) and lungs from mice with pulmonary fibrosis elicited by exposure to bleomycin or silica (24). In these experimental models, pulmonary inflammation and fibrosis were prevented by the injection of anti-TNF- Ab or TNF- antagonist. In both our SEB-induced interstitial pneumonia and bleomycin-induced interstitial pneumonia models, the TNF- mRNA expression was fairly low on Day 14. These results indicate that TNF- may play an important role in the development of interstitial pneumonia, especially in the acute phase after onset. IFN- was released mainly by CD8⁺ T cells in the BALF cells from SEB-treated mice. It enhances the accessory function of antigen-presenting cells (27) and behaves as an antifibrogenic factor with suppressive effects on the production of extracellular proteins (28). However, IFN-'s contribution to the anti-fibrogenic potency in our animal model seems to be small inasmuch as IFN- mRNA decreased at Day 14. It is known that IFN- upregulates the expression of CD95 (29), resulting in an augmentation of apoptosis. The role of IFN- in the bronchoalveolar space is very complicated, and the role of IFN- in apoptosis in the bronchoalveolar space has not been elucidated.

PDGF and insulin-like growth factor (IGF)-I are growth factors for fibroblasts and epithelial cells, and their receptors are abundantly expressed in the lungs of IPF patients (30). Increased expressions of PDGF and IGF-I mRNA in BALF cells from bleomycin-treated mice were observed (33). Although PDGF seems to play an important role in the pulmonary fibrosis in our animal model, the reasons why the increase in PDGF mRNA persists in relatively late phase is not clear. It is necessary to clarify the regulation of the transcription of the PDGF gene in our animal model.

FK506 binds to the intracellular FK-binding protein (34). This complex binds to the serine/threonin-specific phosphatase calcineurin, preventing its activation by calcium and blocking activation of the cytoplasmic nuclear factor of activated T-cell proteins (35, 36). In the present study, FK506 but not corticosteroid blocked clonal expansion of SEB-reactive T cells in BALF and it suppressed TNF-, IFN-, JE/MCP-1, RANTES, and PDGF mRNA expression in BALF cells, resulting in the prevention of the development of interstitial pneumonia in SEB-treated MRL +/+ mice. Although corticosteroids are widely used in the treatment of IPF and other fibrotic lung disorders, they result in favorable clinical response in only one-fourth to one-third of patients with IPF (37), and the effects of corticosteroids have been achieved in only 15 to 30% of cases in pulmonary complications of autoimmune disease (6). In our experiment, corticosteroid treatment had no effect on the mRNA expression of cytokines and chemokines, or on histologic findings. Flow cytometric analysis revealed that the expansion of SEB-reactive T cells in BALF was not inhibited by corticosteroid treatment (data not shown). In in vitro assay, prednisolone was approximately 1,000-fold less effective than FK506 in prevention of SEB-induced proliferation of spleen cells. These data suggest that corticosteroid does not suppress the initiation of SEB-induced inflammation and subsequent induction of interstitial pneumonia. Differences in the actions of FK506 and corticosteroid in this model might be due to different immunologic actions and dosages.

To clarify the role of T cells in this model, we transferred each type of cell of MRL +/+ mice into SCID mice with SEB. Transfer of T cells and SEB was necessary for the development of interstitial pneumonia in SCID mice. These results support the ideas that SEB-induced T-cell expansion may trigger the induction of interstitial pneumonia and that T cells play an important role in this model (although we never exclude the role of other types of cells, such as macrophages and neutrophils).

The mechanism of development of interstitial pneumonia is very complicated. In this study, we demonstrated that SEB-induced T-cell expansion plays an important role in initiation of interstitial pneumonia in our model. Further studies are needed to clarify the mechanisms underlying the development of interstitial pneumonia in this animal model, and the results obtained should contribute to our understanding of human lung fibrosis.