Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets (original) (raw)
Both Th1 cells and Th2 cells contribute to GVHD. To evaluate the separate contributions of Th1 and Th2 cells to the induction of GVHD in a fully MHC-mismatched and multiple minor histocompatibility antigen–mismatched strain combination, we compared the ability of BMC plus spleen cells from BALB/c–backcrossed H-2d_STAT4–/–_ mice, STAT6–/– mice, or WT control BALB/c mice to induce GVHD in lethally irradiated C57BL/6 recipients. The number of donor CD4+ and CD8+ T cells administered in WT, STAT4–/–, and STAT6–/– inocula was similar in all experiments, as measured by flow cytometry analysis of each inoculum. As shown in Figure 1, the most severe mortality occurred in the group receiving WT splenocytes, which are capable of producing both Th1 and Th2 responses (median survival time [MST] = 22 days). STAT6–/– BMT, which is capable of producing high Th1 responses and no Th2 responses, induced mortality (MST = 31.5 days) that was less rapid in onset than that induced by WT BMT (P < 0.01). Mortality caused by the injection of STAT4–/– cells, which lack Th1 and produce enhanced Th2 responses, was consistently less rapid (MST = 79 days) than that caused by both WT (P < 0.0001) and STAT6–/– cells (P < 0.001). Figure 1 shows the mortality of 117 animals from 6 separate experiments. We observed that the rapidity of onset of GVHD varied significantly from experiment to experiment, but in each experiment the GVHD mortality was most rapid when induced by WT cells, followed by STAT6–/– cells and finally STAT4–/– cells. Furthermore, when GVHD was induced in the reverse strain combination, using C57BL/6–backcrossed H-2b_STAT4–/–_, STAT6–/–, or WT control C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients, the same hierarchy of rapidity of GVHD mortality was observed: mortality induced by WT cells was the most rapid, followed by STAT6–/– cells and then STAT4–/– cells, the slowest (data not shown).
Both Th1 cells and Th2 cells contribute to GVHD mortality. Lethally irradiated B6 mice received 5 × 106 T-cell–depleted (TCD) B6 BMC and either syngeneic spleen cells (SYN) (n = 18), BALB/c WT BMC and spleen cells (WT) (n = 38), STAT4–/– (Th2) BMC and spleen cells (n = 31), or STAT6–/– (Th1) BMC and spleen cells (n = 30). Each BALB/c (WT, STAT4–/–, or STAT6–/–) inoculum contained 13 × 106 spleen cells plus 10 × 106 BMC. Pooled data from 6 experiments, involving a total of 117 animals, are shown.
To further demonstrate that both Th1 cells and Th2 cells contribute to acute GVHD, we compared the severity and mortality of GVHD induced by STAT4–/– cells, STAT6–/– cells, and combined STAT4–/– plus STAT6–/– cells. Mice received either a full dose (13 × 106) or a half dose (6.5 × 106) of both STAT4–/– and STAT6–/– spleen cells plus BMC, or 13 × 106 WT, STAT4–/–, or STAT6–/– cells alone. Whereas recipients of the full dose of combined STAT4–/– and STAT6–/– spleen cells developed accelerated GVHD (MST = 6 days) compared with recipients of WT inocula (MST = 16 days), there was no difference in GVHD induced by 13 × 106 WT spleen cells compared with the combination of 6.5 × 106_STAT4–/–_ cells and 6.5 × 106_STAT6–/–_ cells (MST = 15.5 days). Recipients of 13 × 106_STAT6–/–_ or STAT4–/– spleen cells alone showed delayed GVHD compared with recipients of either 13 × 106 WT cells or 6.5 × 106_STAT4–/–_ cells and 6.5 × 106_STAT6–/–_ cells, confirming that both Th1 cells and Th2 cells contribute to acute GVHD.
The addition of STAT4–/– cells to WT cells does not protect against GVHD. It has previously been shown that transplantation of polarized Th2 cells, after 7 days in culture with IL-2 and IL-4, reduced the GVHD mortality caused by nonpolarized cell populations (27). We therefore attempted to determine whether naive Th2 cells could ameliorate GVHD induced by Th1 cells by adding STAT4–/– BMC and spleen cells to WT cells. The addition of STAT4–/– (Th2) cells to WT cells not only failed to prevent or reduce the severity of acute GVHD (MST = 18 days), but it accelerated GVHD mortality compared with that induced by WT cells alone (MST = 31 days) (Figure 2 shows one of two similar experiments). This result further emphasizes that Th2 cells play a pathogenic role in GVHD, and shows that they do not effectively inhibit GVHD mediated by naive cells that can differentiate into Th1 cells.
The addition of STAT4–/– cells to WT BMC plus spleen cell inocula does not protect from GVHD. Lethally irradiated B6 mice received TCD B6 BMC and either syngeneic spleen cells (n = 3), BALB/c WT BMC and spleen cells (WT) (n = 6), STAT4–/– (Th2) BMC and spleen cells (n = 8), or WT BMC and spleen cells plus STAT4–/– BMC and spleen cells (STAT4–/– + WT) (n = 3). Each inoculum contained 13 × 106 BALB/c spleen cells plus 10 × 106 BALB/c BMC (WT-induced control GVHD); 13 × 106 BALB/c STAT4–/– spleen cells plus 10 × 106 BALB/c STAT4–/– BMC (STAT4–/–_-induced GVHD); or 6.5 × 106 BALB/c WT spleen cells plus 6.5 × 106 BALB/c STAT4–/– spleen cells plus 5 × 106 BALB/c WT BMC plus 5 × 106_STAT4–/– BMC (STAT4–/– + WT cells). Results of one of two similar experiments are shown.
Unique clinical and pathological syndromes associated with Th2 and Th1 GVHD. We monitored transplanted animals for clinical signs of GVHD. Figure 3 shows the mortality curve and weight curve from a representative experiment. GVHD scores for generalized signs (hunching, ruffled fur, and periorbital edema) of WT recipients were the most severe in intensity and were the earliest in onset: on day 8, mean GVHD score was 9.8 ± 0.6 out of a possible maximum score of 10. The recipients of STAT6–/– BMT and STAT4–/– BMT had GVHD scores that were lower at this time point (on day 8, mean score for recipients of STAT4–/– BMT was 2.3 ± 1.2, and for recipients of STAT6–/– BMT it was 5.4 ± 0.5) and were similar to each other. However, the time required to achieve the maximal score for generalized signs of GVHD was earlier in recipients of STAT6–/– BMT (on day 12, mean score was 6.7 ± 0.6) than in STAT4–/– BMT recipients (on day 16, mean score was 7 ± 1). A weight curve, which provides a sensitive indicator of GVHD, is shown in the lower panel of Figure 3. Animals receiving WT cells rapidly lost weight before their deaths by day 13. Animals receiving Th1 (STAT6–/–) and Th2 (STAT4–/–) BMT showed initial weight loss during the first 10 days after BMT. However, the clinical signs subsequently diverged markedly in the two groups. Whereas recipients of STAT6–/– BMT developed severe diarrhea and began to show mortality around day 25, recipients of STAT4–/– BMT did not have diarrhea, and had only mild weight loss. As shown in the upper panel of Figure 3, recipients of STAT4–/– BMT began to show mortality around day 30. These mice developed extremely severe skin GVHD, which began around the eyes, mouth, and ears and progressed to involve the whole body, resulting in hair loss and profound scaling. This striking clinical picture was observed in all six experiments, with time of onset ranging from 15 to 50 days after BMT. Whereas these clinical changes were not observed in any experiments in the animals that received BMT from WT or STAT6–/– mice and survived until day 100, they were present in the majority (11 of 14) of STAT4–/– BMT recipients that survived to 10 weeks after BMT.
Weight loss and mortality for WT-, Th1-, and Th2-mediated GVHD in a single representative experiment. GVHD mortality is shown in the upper panel, and GVHD-induced weight loss is shown in the lower panel. Lethally irradiated B6 mice received 5 × 106 TCD B6 BMC and either syngeneic spleen cells (n = 3), BALB/c WT BMC and spleen cells (WT) (n = 6), STAT4–/– (Th2) BMC and spleen cells (n = 8), or STAT6–/– (Th1) BMC and spleen cells (n = 8). Each BALB/c (WT, STAT4–/–, or STAT6–/–) inoculum contained 13 × 106 spleen cells plus 10 × 106 BMC.
Different localization of pathological GVHD-associated changes in recipients of WT, STAT4–/–, and STAT6–/– BMT. After death, carcasses were fixed in formaldehyde and the tissues were processed and scored in a blinded fashion for lesions associated with GVHD. All analyzed organs were harvested from animals that died at similar times after transplantation from two experiments where the severity of GVHD was similar (MSTs in days for experiment 1 and 2: WT = 12 and 19, STAT6–/– = 35 and 29, STAT4–/– = 79 and 155). The liver, spleen, lungs, kidney, and large intestine were examined. There were no pathological changes in the appearance of liver (Figure 4a), skin (Figure 4e), or large intestine (Figure 4i) in recipients of syngeneic BMT. Moderate to severe GVHD-induced liver changes were observed in 5 of 5 recipients of WT BMT, which died on days 19, 24, 27, and 54 after BMT. Figure 4b shows typical liver lesions found in these mice; portal areas were diffusely infiltrated by a mixed population of leukocytes including neutrophils, lymphocytes, macrophages, and a few plasma cells. There was associated multifocal, single-cell hepatocellular necrosis, consistent with severe hepatic GVHD. Similar changes were observed in 4 of 4 recipients of STAT4–/– BMT, which died on days 20, 39, 47, and 106 after BMT (Figure 4d). In striking contrast, liver sections of STAT6–/– BMT recipients that died at similar times (22, 23, 25, 26, 27, 29, and 38 days after BMT) appeared normal, without any infiltrate or evidence of cholestasis (Figure 4c).
Histological evidence of GVHD in the liver, skin, and large intestine. Liver, skin, and large intestine sections were obtained from lethally irradiated B6 recipients reconstituted with syngeneic BMT (a, e, i), WT BMT (b, f, j), STAT6–/– BMT (c, g, k), or STAT4–/– BMT (d, h, l). Lethally irradiated B6 mice received 5 × 106 TCD B6 BMC and either syngeneic spleen cells, BALB/c WT BMC and spleen cells (WT), STAT4–/– (Th2) BMC and spleen cells, or STAT6–/– (Th1) BMC and spleen cells. Each BALB/c (WT, STAT4–/–, or STAT6–/–) inoculum contained 13 × 106 spleen cells plus 10 × 106 BMC. These samples, with the exception of those from recipients of syngeneic BMT, were harvested from animals that died during the third week after BMT from two experiments in which the severity of GVHD was similar. Animals that died at later time points showed similar pathological changes. Syngeneic BMT recipients were sacrificed 200 days after BMT. Photographs were taken through a ×50 objective lens.
All recipients of WT BMT showed signs of slight skin hyperkeratosis. In some mice (3 of 7) that died on days 19 and 27 after BMT, there were very mild focal mononuclear inflammatory cell infiltrates located mostly in the dermis (Figure 4f). In accordance with the clinical picture, recipients of STAT4–/– BMT (3 of 4) that died on days 20, 39, and 106 after BMT showed marked skin abnormalities. In some animals, pustules and crusting with neutrophil infiltration was observed on the skin’s surface (Figure 4h). In all animals, the dermis was infiltrated by a mixed population of leukocytes, including lymphocytes, macrophages, and neutrophils (Figure 4h). In several places the cells formed granulomas. There was also a suggestion of lysis along the epidermal-dermal junction. Consistent with the clinical picture, inflammatory changes were absent in all 12 recipients of STAT6–/– BMT that died on days 5, 22, 23, 25, 26, 27, 29, 38, and 44 after BMT (Figure 4g). Furthermore, these severe skin changes were not observed in mice with GVHD induced by STAT6–/– that had long-term survival (4 animals that died at 74, 122, 149, and 150 days after BMT). Their skin showed signs of mild hyperkeratosis, but without inflammatory changes.
Recipients of WT and STAT6–/– BMT showed clinical signs of severe colitis manifested by profuse diarrhea and weight loss. The pathological examination revealed that in 7 of 7 recipients of WT inocula, which died on days 19, 24, 27, 39, and 54 after BMT, there was a thickening of colonic mucosa with mild to severe infiltration of mononuclear inflammatory cells. The epithelium was hyperplastic with elongated, tortuous, and branched crypts (Figure 4j). The colon sections of recipients of STAT6–/– BMT showed similar changes in 9 of 9 examined animals, which died on days 5, 22, 23, 25, 26, 27, 29, 38, and 44 after BMT. Three of 9 animals had extremely severe chronic colitis, with lumens that were filled with exudate and severely ulcerated mucosa (Figure 4k). The changes observed in recipients of STAT6–/– BMT were more severe than changes observed in recipients of WT BMT.
Although the recipients of STAT4–/– BMT lacked clinical signs of colitis or weight loss, in 3 of 4 animals that died on days 39, 47, and 106 after BMT, there were histological signs of colitis, with a thickening of the mucosa and diffuse cellular infiltrate (Figure 4l). However, these changes were less severe than those observed in recipients of WT or STAT6–/– BMT.
There were no pathological changes in the appearance of lungs or kidneys in any of the 3 groups of recipients (not shown).
STAT6–/– cells and STAT4–/– cells exhibit stable Th1 and Th2 phenotypes, respectively, in vivo. Cytokines modulate B-cell function by differentially stimulating specific Ig isotype production. Th1 responses are characterized by an increase in the level of IgG2a production (2). Similarly, immune responses characterized by high levels of IL-4 lead to elevated serum IgE and IgG1, and suppress the production of IgG2a (2). Increases in serum IgE have been reported in GVHD in man and mouse (28, 29). We followed serum IgE levels in the GVHD mice over time. Normal nontransplanted C57BL/6 and BALB/c age- and sex-matched mice contained an average of 300 ng/mL IgE in their sera at the age of 20 weeks. IgE levels in sera of the WT and syngeneic BMT recipients did not differ significantly from those of controls (Figure 5a). In contrast, the recipients of STAT4–/– BMT–induced GVHD showed significantly increased serum IgE levels as early as 2 weeks after BMT (Figure 5a). These levels increased over time, becoming greater than 4,000 ng/mL in GVHD mice sacrificed 5 months after BMT. The recipients of STAT6–/– BMT showed almost no detectable IgE in the serum at the time of sacrifice 5 months after BMT (Figure 5a). This profile of high levels of IgE in STAT4–/– BMT–induced GVHD, and IgE deficiency in the recipients of STAT6–/– BMT, indicates that donor-derived T cells retain their Th1 or Th2 phenotype in GVHD mice.
Serum IgE (a) and IgG1 (b) levels in GVHD induced by WT, STAT6–/–, and STAT4–/– cells. C57BL/6 animals were transplanted with syngeneic, WT BALB/c, STAT4–/–, or STAT6–/– cells. Lethally irradiated B6 mice received 5 × 106 TCD B6 BMC and either syngeneic spleen cells, BALB/c WT BMC and spleen cells (WT), STAT4–/– (Th2) BMC and spleen cells, or STAT6–/– (Th1) BMC and spleen cells. Each BALB/c (WT, STAT4–/–, or STAT6–/–) inoculum contained 13 × 106 spleen cells plus 10 × 106 BMC. For syngeneic BMT and for WT- and _STAT6–/–_-induced GVHD, animals were sacrificed at 5 months after BMT. Recipients of STAT4–/– cells were bled and sacrificed at 2 weeks, at 3 months, and at 5 months after BMT. Sera were collected and Ig levels were measured by ELISA. For each time point for each group, 2–4 animals were analyzed. MSTs in days for this experiment were 26 for recipients of WT cells, 54 for recipients of STAT4–/– cells, and 35 for recipients of STAT6–/– cells. Concentrations of IgE and IgG1 in sera are expressed as mean ± SEM.
We also followed the IgG1 levels in the recipients of STAT4–/–, STAT6–/–, WT, and syngeneic BMT. The levels of IgG1 in sera of these animals followed the IgE pattern. The recipients of STAT4–/– GVHD inocula had significantly higher levels of IgG1 in their sera than did the recipients of WT, STAT6–/–, or syngeneic inocula, and this level increased over time (Figure 5b). In contrast, the recipients of STAT6–/– GVHD inocula had almost undetectable levels of serum IgG1 (Figure 5b). Total IgG levels were similar in the recipients of STAT6–/– BMT (312.9 ± 152.0 μg/mL) sacrificed at 5 months after BMT and in recipients of STAT4–/– cells (470.3 ± 38.0 μg/mL) sacrificed at 2 weeks after BMT, despite the difference in IgG1 levels cited above.
Donor engraftment and T helper phenotype in recipients of WT, STAT4–/–, and STAT6–/– BMT. Recipients of STAT4–/– cells were sacrificed at 2 weeks, 3 months (87 days), and 5 months (136 days) after BMT. Recipients of syngeneic and WT cells were sacrificed at 2 weeks and at 3 months (87 days) after BMT. For _STAT6–/–_-induced GVHD, animals were sacrificed only at 5 months (136 days) after BMT. MLCs were prepared with host-type (B6) irradiated stimulator cells. FACS® analysis of peripheral blood lymphocytes and bone marrow showed that these animals were full donor-type hematopoietic chimeras at all 3 time points (data not shown). The splenocytes from these animals showed mixed lymphocyte response reactivity both toward host C57BL/6 cells and third-party strain A/J, while being nonreactive toward donor antigens (data not shown). Thus, there was constant, ongoing activation and proliferation of donor T cells toward host antigens. By quantifying the levels of IFN-γ, IL-2, IL-4, and IL-10 in MLC supernatants from WT and STAT4–/– BMT recipients, distinct patterns emerged. In supernatants obtained from spleen cells of mice receiving STAT4-deficient cells, the amounts of IL-4 and IL-10 (i.e., the Th2-specific cytokines) were increased compared with those in supernatants of spleen cells from WT recipients (see Figure 6). Similar results were obtained at all 3 time points after BMT. IFN-γ and IL-2, which are both induced during the Th1 response, were detected at higher levels in supernatants of spleen cells from recipients of WT BMT than in recipients of STAT4–/– BMT (see Figure 6). Although produced at lower levels than in the recipients of WT BMT, there was measurable production of IFN-γ in the recipients of STAT4–/– BMT (see Figure 6). Due to profound atrophy of the spleen at 5 months after BMT, only 1 recipient of STAT6–/– BMT was available for analysis. This mouse showed the expected Th1 phenotype, with large amounts of IFN-γ (781 pg/mL) and IL-2 (591 pg/mL), low amounts of IL-10 (228 pg/mL), and undetectable IL-4 in the collected supernatants.
Cytokine profile of splenocytes from mice with WT- or STAT4–/–_-induced GVHD. Lethally irradiated C57BL/6 host mice transplanted with 5 × 106 TCD B6 BMC and either 13 × 106 WT spleen cells plus 10 × 106 WT BMC (n = 2) or 13 × 106_STAT4–/– spleen cells plus 10 × 106_STAT4–/–_ BMC (n = 4) were sacrificed at 2 weeks after BMT, and splenocyte suspensions were prepared. Their donor-derived phenotype was demonstrated by FACS®. The cells were cultured with irradiated splenocytes from C57BL/6 mice. Supernatants were harvested 72 hours after initiation of MLC, and cytokine levels were determined by ELISA. MSTs for this experiment were 26 days for recipients of WT cells and 54 days for recipients of STAT4–/– cells. Concentrations of IFN-γ, IL-2, IL-4, and IL-10 in cell culture supernatants are expressed as mean ± SEM (pg/mL). A_P_ < 0.01 vs. WT group.