SEARCH

SEARCH BY CITATION

Keywords:

  • Intestinal intraepi-thelial lymphocyte;
  • Mesenteric lymph node lymphocyte;
  • TNF-α

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods

The gastrointestinal tract is a major target of graft-versus-host disease (GVHD), which constitutes a life-threatening complication of bone marrow transplantation. GVHD is mainly caused by the activation of donor-derived lymphocytes, in which cytokine cascades play essential roles. Since p38 MAPK (p38) has been identified as a regulator of cytokine reactions and proposed as a molecular target for anti-inflammatory therapy, we investigated the contribution of p38 to the severity of murine intestinal GVHD. Unexpectedly, p38α+/− donor graft induced more acute GVHD-related mortality and more severe gut injury. The survival of p38α+/− donor-derived intestinal intraepithelial lymphocytes (IEL) was prolonged in vitro and in vivo, and TNF-α expression in the p38α+/− donor-derived IEL was also increased compared with wild-type cells. In contrast, the p38α+/− grafted mice resulted in decreased expansion of donor lymphocytes in mesenteric lymph nodes, and the up-regulation of IL-12p40 and IL-18 was diminished. These findings suggest that p38 has dichotomous effects for inflammatory response invivo; not only regulates inflammatory cytokine expression and lymphocyte expansion, but also has distinct regulatory functions for IEL in intestinal GVHD. In conclusion, the inhibition of p38 may not be a suitable anti-inflammatory strategy for GVHD due to the associated intestinal injury.

Abbreviations:
BMT:

Bone marrow transplantation

B6:

C57BL/6

IEL:

Intestinal intraepithelial lymphocytes

MLNL:

Mesenteric lymph node lymphocytes

SPC:

Splenocytes

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods

Allogeneic bone marrow transplantation (BMT) is recognized as an indispensable therapy for a number of malignant diseases. However, the benefits of BMT are often canceled by serious complications, the most common of which is GVHD. The most frequent target organs of GVHD are the gastrointestinal tract, liver, and skin 1, 2. Most importantly, gastrointestinal injury is a major cause of morbidity and mortality, with profuse diarrhea, ileus, and in some cases, bleeding 3, 4. Although the mechanism of intestinal GVHD is complex, this disease is considered to be the result of host antigen-mediated donor T cell responses, which are activated by pro-inflammatory cytokines and endotoxins 4, 5. Several studies have suggested that intestinal lesions are initiated by the activation of donor T cells by allogeneic antigen presentation, while the disease is exacerbated by cytokine release from the activated T cells 6, 7.

The MAPK have essential roles in the integration and processing of various extracellular signals. In particular, p38 MAPK (p38) is activated by cytokines and stress during various inflammatory responses via MAPK kinase (MKK)3, MKK4, and MKK6 8, 9. Among the four mammalian isoforms of p38 (p38α, -β, -γ, and –δ), p38α is expressed relatively ubiquitously 1012, and p38α-deficient mice have been reported to be homozygous embryonic lethal due to either abnormal erythropoiesis or placental organogenesis 13. The p38 plays a pivotal role in Th1/Th2 responses by regulating the expression of cytokines, such as IL-2, IL-4, IL-12, and IFN-γ 1417, and also plays important roles in modulating proliferation and cell death 1821.

Because of its crucial role as a key regulator of inflammatory process, p38 has been targeted for anti-inflammatory therapy, e.g. p38-specific inhibitors have been used in various animal models of inflammatory disease 2224. However, there are contradictory reports on the effectiveness of inhibiting p38 in experimental colitis 25 and in an infectious disease model. Although GVHD is also an inflammatory disease involving various cytokines 6, 26, the function of the p38 in intestinal GVHD has not been explored. In this report, we established the experimental model using p38 gene-targeted mice 13, 27 and analyzed the pathological role of p38 in acute intestinal GVHD.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods

Reduced p38α in donor cells induces more severe GVHD phenotypes in recipient mice

We used a well-established parent to F1 murine model for allogeneic BMT/donor lymphocyte infusion (DLI) to study acute GVHD. In these experiments, we used WT or p38α+/− donor mice to determine whether the p38α activities of the donor graft contributed to acute GVHD. Initially, survival periods and body weights, which are reliable markers of the systemic severity of GVHD 4, were analyzed in the WT and p38α+/− donor-infused recipients. The transplantation of 3 × 107 splenocytes (SPC) from B6-background mice into irradiated BDF1 mice resulted in 100% mortality for the allo-donor-grafted mice, whereas the syngeneic-grafted mice showed neither GVHD-related death nor body weight loss, even after lethal-dose irradiation and transplantation. (Fig. 1A). Considering the pro-inflammatory polarity of the p38α-mediated signal, it was somewhat surprising to discover that the mortality rate was significantly higher in the Allo-p38α+/−-grafted mice than in the Allo-WT-grafted mice (P < 0.001; Fig. 1A). The Allo-p38α+/−-grafted mice also showed significantly higher weight loss than the Allo-WT-grafted mice (Fig. 1B) and more severe systemic GVHD assessed by clinical score at 21 days after transplantation (Fig. 1C, p < 0.01). To determine whether the clinical phenotypes were caused mainly by the transfer of allogeneic, donor-derived T cells, mice that underwent BMT without splenocytes (spc) (Allo-WTspc− and -p38α+/− spc−) or BMT with purified T cells were also prepared in the allograft groups (Allo-WTtcell+ and -p38α+/− tcell+). Lethal GVHD was only induced in mice transplanted with allogeneic T cells, and the mortality rate was also significantly higher in mice transplanted with Allo-p38α+/− T cells (log-rank test, p < 0.05; Fig. 1D). These mice also showed significantly greater weight loss (data not shown) and more severe systemic GVHD at 21 days after transplantation (p < 0.05, Fig. 1C) than did the Allo-WT T cell-transplanted mice.

thumbnail image

Figure 1. Evaluation of the level of GVHD for each of the following grafted groups: syngeneic B6 recipients grafted from GFP-WT donors (Syn-WT); B6 recipients grafted from GFP-p38α+/− donors (Syn-p38α+/−); allogeneic BDF1 recipients grafted from GFP-WT donors (Allo-WT); and BDF1 recipients grafted from GFP-p38α+/− donors (Allo-p38α+/−). Mice that underwent BMT without SPC transplantation (Allo-WTspc− and -p38α+/− spc−), or BMT with purified T cell transferred were also prepared in the allo-grafted groups (Allo-WTtcell+ and -p38α+/− tcell+). (A) The post-transplantation mortality rates for the indicated group ( n= 14, respectively) were monitored daily in three independent experiments. (B) Body weight changes are shown as percentages of the day 0 weight for three independent experiments using GVHD-induced mice (Allo-WT and -p38α+/−; n = 18) and syngeneic-grafted control mice (Syn-WT, and -p38α+/−; n = 6). (C) The systemic GVHD severity of GVHD-induced mice groups (Allo-WT and -p38α+/−; n=6; Allo-WTtcell+ and -p38α+/− tcell+, n = 3) and control grafted mice (Syn-WT, -p38α+/−; n = 4, Allo-WTspc− and -p38α+/− spc−, n = 3) was analyzed by the clinical scoring system on day 21 after transplantation. (D) Monitoring of post-transplantation mortality rates for the GVHD model transplanted with allogeneic T cells (n = 6). *** p < 0.001, ** p <0.01, and * p< 0.05 vs. Allo-WT-grafted mice.

Download figure to PowerPoint

Pathological findings of intestinal GVHD are more severe in Allo-p38α+/−-grafted mice

The pathological changes in the bowel tissues were examined to assess intestinal injury caused by GVHD. At day 21 post-transplantation, severe gut injuries were observed in both groups of Allo-WT-grafted mice (Fig. 2C, D) and Allo-p38α+/−-grafted mice (Fig. 2E, F), in contrast to the syngeneic-grafted mice (Fig. 2A, B), which did not show significant changes in the mucosal architecture of the gut tissues. Most notably in the Allo-p38α+/−-grafted mice, the typical histological features of intestinal GVHD 4, i.e. marked villous blunting, lamina propria lymphocytic and granulocytic infiltration (Fig. 2E), and crypt destruction (Fig. 2F), were evident. Although these features were also noted in Allo-WT-grafted mice (Fig. 2C, D), the histological severity was mild compared with the Allo-p38α+/−-grafted mice.

thumbnail image

Figure 2. Bowel sections from syngeneic-WT-grafted mice (A, B), Allo-WT-grafted mice (C, D), and Allo-p38α+/−-grafted mice (E, F) were examined histologically on day 21 post-grafting. Representative photomicrographs are shown for the six mice examined from each group.

Download figure to PowerPoint

Reduced p38α activities in donor lymphocytes increase TNF-α concentration and the single-cell apoptosis in the intestinal tissue

The presence of epithelial single-cell apoptosis in gastrointestinal tissues is one of the hallmarks of gastrointestinal GVHD 28. To confirm the severity of gut GVHD, the number of apoptotic cells in the colonic tissues were analyzed by the TUNEL assay. Many apoptotic bodies were found in the GVHD-induced gut tissues (Fig. 3B, C), in contrast to the gut tissues of syngeneic-grafted mice (Fig. 3A), and the number of apoptotic bodies was significantly increased in Allo-p38α+/−-grafted mice, as compared to Allo-WT-grafted mice (Fig. 3D). TNF-α is one of the most well known cytokines that promotes various inflammatory responses and is implicated in the severity and mortality of GVHD 4, 6. The TNF-α concentrations in the colon homogenates analyzed by ELISA on day 21 were elevated in both groups of GVHD-induced mice but not in the syngeneic-grafted mice, while the TNF-α levels were significantly increased in the Allo-p38α+/−-grafted mice, as compared to the Allo-WT-grafted mice (Fig. 3E).

thumbnail image

Figure 3. TUNEL staining of the intestinal mucosa of syngeneic-WT- (Syn-WT; A), allogeneic-WT- (Allo-WT; B), and allogeneic-p38α+/−- (Allo-p38α+/−; C) grafted mice on day 21 post-transplantation. Numerous apoptotic bodies (arrowhead) are found in the Allo-p38α+/−-grafted mice, as compared to the Allo-WT-grafted mice. Representative photomicrographs of each group are shown. Original magnification: (A–C) ×200. (D) The TUNEL-positive cells were counted (black spots) per 100 crypts of Allo-WT- (n = 6), Allo-p38α+/−- (n = 6), and Syn-WT-grafted mice (n = 4). The bars indicate the mean ± SEM numbers of apoptotic cells per 100 colonic crypts. (E) The TNF-α concentrations in the bowel tissue homogenates from GVHD-induced and syngeneic-control mice were measured by ELISA on day 21 after transplantation. ** p < 0.01; * p <0.05; and N.S., not significant.

Download figure to PowerPoint

Analysis of donor-derived IEL/MLNL kinetics in GVHD mice

Donor-derived intestinal intraepithelial lymphocytes (IEL) infiltration is considered to be the major cause of host intestinal injury, based on the strong cytotoxic capability of IEL in intestinal GVHD 29. Marked expansion of the GFP-labeled donor-derived lymphocytes was observed in the gut tissues of GVHD mice, but not in the corresponding tissues of syngeneic-grafted mice (Fig. 4A, B). To address the effector kinetics of allogeneic reactions in the intestine, the numbers and fraction profiles of these donor-derived IEL and mesenteric lymph node lymphocytes (MLNL) were examined by flow cytometry. At day 12 post-transplantation, the GFP-labeled donor-derived lymphocytes had already infiltrated into the intra-epithelia of the host intestinal tissue (Fig. 4C). It was also confirmed by flow cytometry that the isolated IEL of both allo-grafted mice groups were derived primarily from donors, and composed mainly of CD8+ lymphocytes distinct from MLNL already at 12 days after transplantation (Fig. 5, Table 1). Analysis of donor-derived IEL/MLNL kinetics in GVHD mice revealed that both the infiltration of donor-derived IEL and MLNL peaked on day 12, and declined thereafter in the Allo-WT-grafted mice (Fig. 6A, B). Meanwhile, in the Allo-p38α+/−-grafted mice, although the donor-derived MLNL were decreased compared to the Allo-WT-grafted mice, the IEL infiltration still persisted after 12 days, relatively, and significantly increased at day 21 compared to the Allo-WT-grafted mice (p < 0.02). The IEL and MLNL from syngeneic-grafted mice were also analyzed, and it was confirmed that p38α had no influence on the expansion of donor-derived lymphocytes in syngeneic bone-marrow-replaced mice (data not shown), indicating that there was no influence of the bone marrow cells (BMC) reconstruction ability between the WT and p38α+/− donor mice. The differences in donor-derived lymphocyte kinetics observed in Allo-grafted GVHD mice probably reflect the altered graft-versus-host reaction brought about by decreased p38α.

thumbnail image

Figure 4. Identification of donor lymphocytes that have infiltrated the intestinal tissues of Syn-WT- (A) and Allo-WT-grafted mice (B) on day 12 post-transplantation. Donor-derived IEL of the intestinal crypt of the Allo-WT-grafted mice (C, white arrows). Green color indicates GFP positive donor-derived cells; red color indicates PI staining for DNA. Bar: (A, B), 200 μm; (C), 40 μm.

Download figure to PowerPoint

thumbnail image

Figure 5. On day 12 post-transplantation, the MLNL and IEL were isolated from Allo-WT- and -p38α+/−-grafted mice (n = 6). The GFP positive donor-derived cells occupied over 95% of collected MLNL and IEL both in the groups (left panels). The proportions (%) of the CD4 or CD8 positive lymphocytes of the GFP-positive cells were analyzed (each right panel). Representative data from each of the two recipient groups are shown.

Download figure to PowerPoint

Table 1. Total cell numbers and CD8/CD4 population of donor-derived IEL and MLNL at day 12 post-transplantation in GVHD induced micea)
GroupsIsolated cellsTotal cell number (×106)%Donor CD8+%Donor CD4+
  1. a) IEL and MLNL were isolated from the both GVHD-induced mice groups (Allo-WT and - p38α+/−) on day 12 post-transplantation, and the total cell number and donor-derived CD8/CD4 population were assessed by flow cytometry.b) p < 0.01; c) p < 0.05; and N.S., not significant vs. Allo-WT-grafted mice.

Allo-WTIEL9.88 ± 1.8270.6 ± 2.211.4 ± 2.7
Allo-p38α+/−IEL5.48 ± 0.63b)67.4 ± 6.2N.S.11.1 ± 2.5N.S.
Allo-WTMLNL2.22 ± 0.4450.5 ± 8.334.1 ± 2.9
Allo-p38α+/−MLNL1.24 ± 0.34b)39.3 ± 4.1c)39.9 ± 3.1N.S.
thumbnail image

Figure 6. The numbers of donor-derived IEL (A) and MLNL (B) isolated from Allo-WT- and -p38α+/−-grafted mice on days 7 (n = 9), 12 (n = 6), and 21 (n = 6) for two or three independent experiments by flow cytometric analysis. ** p < 0.01 and * p < 0.05 vs. Allo-WT-grafted mice.

Download figure to PowerPoint

The p38α regulate cytokine expression by MLNL and IEL

Since both the donor lymphocyte activation and expansion to target organs are linked to various cytokine stimulations 30, the mesenteric lymph nodes were isolated from Allo-WT- and Allo-p38α+/−-grafted mice on day 12 post-BMT, and the levels of cytokine expression were estimated by quantitative RT-PCR. The expression of IFN-γ, which is a potent Th1 cytokine inducer, was clearly up-regulated in the Allo-grafted mice, whereas the expression of IL-4, which is a master regulator of Th2 immune responses, was down-regulated in the GVHD mice (Fig. 7A, B). A reduction in the level of p38α also caused a reduction in IL-4 gene expression (Fig. 7B), while the expression of IFN-γ was not affected significantly. In addition, the expression of IL-12p40 and IL-18 in MLNL was up-regulated in the Allo-WT-grafted mice, but the increase was inhibited in the Allo-p38α+/−-grafted mice (Fig. 7C, D). On the other hand, TNF-α gene expression in IEL isolated from GVHD mice increased with GVHD progression (Fig. 7E). Interestingly, in comparison with the Allo-WT-grafted mice, the Allo- p38α+/−-derived IEL expressed high levels of TNF-α 12 days after transplantation.

thumbnail image

Figure 7. Cytokine gene expression in the mesenteric lymph nodes isolated from GVHD mice was estimated by quantitative RT-PCR for IFN-γ (A), IL-4 (B), IL-12p40 (C), and IL-18 (D). The expression level was normalized in relation to the HPRT internal control. The mesenteric lymph nodes were isolated from Allo-WT- (black bar) and Allo-p38α+/−- (open bar) grafted mice on day 12 (n = 6) after GVHD induction. Those isolated from untreated mice were also examined (n = 6), and are described as the day 0 controls. (E) The IEL were isolated from Allo-WT- and -p38α+/−-grafted mice on days 7, 12, and 21 (n = 4) after GVHD induction. The filled bar represents the Allo-WT-grafted mice, the open bar represents the Allo-p38α+/−-grafted mice. *** P < 0.001, ** P < 0.01, * P < 0.05 and N.S., not significant vs. Allo-WT-grafted mice.

Download figure to PowerPoint

The p38α contributes to the lifespan of donor-derived IEL in vitro

The reason for the sustained infiltration of IEL at day 21 remains unclear. We isolated donor-derived IEL from GVHD mice at day 21 and examined the viability of the IEL invitro. Isolated IEL from GVHD-induced mice were cultured, and some of the lymphocytes were stimulated by pre-coating with purified anti-TCR-β Ab (10 μg/ml) and soluble anti-CD28 Ab (0.5 μg/mL). The number of viable cells was determined by trypan blue staining after 48 h of incubation. Strikingly, the survival time of the Allo-p38α+/−-derived IEL was longer than that of the Allo-WT-derived IEL, regardless of whether or not they were costimulated by TCR/CD28 (Fig. 8A). In addition, lactose dehydrogenase (LDH) release assay also showed that the level of LDH in the Allo-p38α+/−-derived IEL culture medium was less than that in the Allo-WT-derived IEL culture, which was consistent with the results of the viability assay (Fig. 8B).

thumbnail image

Figure 8. The viability of isolated IEL from GVHD mice was assayed by trypan blue staining (A) and the lactose dehydrogenase (LDH) release cell death assay (B). At day 21, donor-derived IEL were isolated from Allo-WT- and -p38α+/−-grafted mice (n = 6) and cultured in the indicated medium at a concentration of 5 × 105/mL, with or without TCR/CD28 stimulation. * p <0.05 and N.S., not significant vs. Allo-WT-grafted mice.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods

Our results show that a reduction in the p38α activity of the donor T cell increases the morbidity and mortality of GVHD-induced mice and the pathological severity of intestinal GVHD, which is followed by an increase in the number of donor CD8+ lymphocytes in the gut intra-epithelia. Meanwhile, in the mesenteric lymph nodes, where pre-matured T cells expand and mature into cytotoxic T cells, donor-derived CD8+ and CD4+ lymphocytes are fewer in p38α+/−-grafted mice. Several studies have reported that activated p38 up-regulates the expression of several cytokines, such as IL-12p40, IL-18, and IL-4 16, 17, 27, and plays essential roles in T cell proliferation in vitro and in vivo1820. The induction of Th1-dominant cytokines, such as IL-12p40 and IL-18, was suppressed in the MLNL of p38α+/−-grafted mice, which may be one of the reasons why fewer lymphocytes infiltrated into the MLNL. Although IFN-γ up-regulation was maintained in the p38α+/−-grafted mice, Th1 differentiation by IL-12 may have been reduced by the decreased p38 activity, as reported previously 31. Moreover, since IL-12 is considered to be essential for the viability of Th1 CD4+ T cells 3235, the expansion of activated CD4+ T cells may be suppressed in p38α+/−-grafted mice. If this is true, then it is interesting to note the discrepancy between the numbers of expanded donor-derived IEL and MLNL. Surprisingly, the life span of IEL in the in vitro cultures was significantly longer when p38 was deactivated. Previous studies show that p38 has an essential role in cell survival via regulation of apoptotic signals in CD8+ but not in CD4+ lymphocytes, although p38 is similarly activated in both types of lymphocytes 21. In intestinal GVHD, donor-derived cytotoxic CD8+ T cells selectively assembled within the host intestinal epithelium (Fig. 5), suggesting that low activation of p38 in p38α+/− lymphocytes prolongs the survival of donor-derived activated IEL, and these phenomena could partly explain the increase in IEL number, as well as the severity of intestinal injury caused by the reduced levels of p38. Since the activation of p38 by TCR/CD28 costimulation did not affect the viability of IEL in these assays, the difference in survival rates does not appear to be linked to p38 activation by TCR stimulation. These data indicate the dichotomous role of p38 in inflammatory responses in vivo; while p38 regulates the expression of key inflammatory cytokines and associated lymphocyte expansion, it may also regulate anti-survival signals in lymphocytes. In other words, p38 may have the potential to attenuate inflammation by eliminating the survival signals of activated lymphocytes within the inflammatory lesion.

TNF-α, which is an important pro-inflammatory cytokine, contributes to T cell activation and differentiation in GVHD, and directly amplifies organ injury 36. Treatment with an anti-TNF antibody has been shown to ameliorate murine GVHD 6, 37. Since the level of TNF-α was higher in the gut tissues of Allo-p38α+/−-grafted GVHD mice than in Allo-WT-grafted mice, it is possible that increased levels of TNF-α accelerate intestinal injury in p38α+/−-grafted mice. Our experiments indicate that TNF-α gene expression is also elevated in Allo-p38α+/−-derived IEL. It has been reported that TNF-α is regulated by p38 in monocytes 38, although contradictory results have also been reported 39. On the other hand, TNF-α production is not only dependent on p38 but is also correlated with extracellular signal-regulated kinases (ERK) in T cells 40. Moreover, it has been reported that CD3/CD28-costimulated lymphocytes produce higher amounts of TNF-α through p38 inhibition in murine experimental colitis 25. On the basis of these findings, it seems likely that p38 inhibition does not always reduce TNF-α production by T cells. Indeed, p38 inhibition may increase TNF-α production by some cell types, such as IEL, or under certain conditions.

The gastrointestinal bacterial product LPS is a potent stimulator of pro-inflammatory cytokines, including TNF-α, and is associated with increased severity of GVHD 4, 5. It has been reported that p38 inhibition results in increased production of cytokines, such as TNF-α, accompanied by severely reduced bacterial clearance in a murine infectious disease model 39. Furthermore, recent evidence suggests that the phagocytotic gene programs of macrophage are regulated by MAPK 41. We analyzed the expression of mRNA for TLR-2, -4 and -9 in the macrophages of mesenteric lymph nodes in both WT and p38α+/−-transplanted GVHD mice, but there were no differences in those expressions between the groups, and we were also unable to detect significant amounts of LPS in the sera of any of the grafted mice 21 days after GVHD induction (data not shown). Nevertheless, it is possible that the reduced level of p38 may have down-regulated the protective immunity, thereby permitting bacterial outbreak in the gastrointestinal tissues that were already injured by the graft-versus-host reaction. This would lead to the production of large amounts of TNF-α and other pro-inflammatory cytokines in the host bowel tissue, which would exacerbate gut inflammation.

The present study indicates that p38 has a broader role in intestinal immune responses, both in its active and inactive forms. It is anticipated that future studies will elucidate the role of p38 in inflammatory process in vivo, and provide other valuable insights into the potential of p38 as a therapeutic target for anti-inflammatory therapies.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods

Animals

C57BL/6 (B6 [H-2b]) and B6D2 F1 (BDF1 [H-2b/d]) mice were obtained as recipient mice from CLEA Japan (Tokyo, Japan). The B6 mice that carry the GFP transgene (B6-GFP mice) were a gift from Dr. Masaru Okabe (Osaka University, Osaka, Japan) 42. Mice heterozygous for targeted disruption of the p38α gene in the B6 background (p38α+/−) were maintained in RIKEN BioResource Center (Tsukuba, Japan) 13, 27. GFP-WT and GFP-p38α+/− mice were generated as donor mice by mating B6-GFP and B6-p38α+/− mice. All procedures involving experimental animals were performed in accordance with protocols approved by the institutional committee for animal research of The University of Tokyo and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86–23, revised 1985).

Transplantation

The well-established parent-F1 hybrid model was used for the induction of graft-versus-host reactions 4. On the day of transplantation, the age-matched (8- to 10-week-old) female BDF1 and B6 mice were irradiated (12.0 Gy; MBR-1520RB, Hitachi, Japan), and injected 3 h later with donor graft cells. Briefly, 3 × 107 SPC and 3 × 106 BMC from GFP-WT or GFP-p38α+/− donor mice were injected via the lateral tail vein into the allogeneic BDF1 mice and syngeneic B6 mice. In some cases, BMC alone, or BMC and purified T cells (3 × 106), were transplanted. Total T cells were isolated from SPC donor mice (GFP-WT or GFP-p38α+/−) by magnetic cell sorting (MACS pan T cell isolation kit, Miltenyi Biotec, Bergisch-Gladbach, Germany); the isolated T cells contained those with a phenotype compatible with regulatory T cells (CD25+, CD45RA+). The purity and recovery of isolated T cells were examined by flow cytometry (recovery > 70%, purity > 95%). It was confirmed that the total SPC number and the populations of CD4+ and CD8+ lymphocytes in p38α+/− mice did not differ from the age- and sex-matched WT mice. The p38 expression was reduced by half in purified T cell populations as well as in total SPC of p38α+/− mice, and the p38 phosphorylation was also reduced by half in p38α+/− SPC with either anti-TCR plus anti-CD28 antibodies [immobilized anti-TCR (10 μg/ml) and anti-CD28 (2.5 μg/ml), stimulated for 10 min] or LPS (10 μg/ml, stimulated for 10 min) (data not shown).

Clinical and pathological assessment of intestinal GVHD

Survival was monitored daily, and body weight was measured every other day. The baseline body weight was determined just after transplantation. To avoid bias from cage-related effects, the animals were randomized before and after transplantation.

On day 21 after transplantation, the degree of systemic GVHD was assessed by a modified scoring system previously described 4. For stool consistency, 0 points were assigned for well-formed pellets, 1 point for pasty stools, and 2 points for liquid stools. A clinical index was subsequently generated by summation of the six criteria scores (maximum index = 12).

To assess pathological features of gut tissues in GVHD, paraformaldehyde-preserved large bowel specimens were stained with hematoxylin and eosin. In order to detect apoptotic cells in the gut tissue sections, a modified in vivo TUNEL staining was performed using the ApopTag Plus Peroxidase Kit (Chemicon Inc., Temecula, California, USA), as described previously 43. The number of apoptotic cells was counted for 100 colonic crypts in four different views. All of the slides were coded and examined in a blinded fashion.

Identification of donor-derived lymphocytes that infiltrate the gut tissue

To preserve the GFP signals of the donor-derived lymphocytes for histological analyses, the intestinal tissues were embedded using the Technovit Catalyst System (Heraeus Kulzer, Wehrheim, Germany) 44. The sections were stained with 1 μg/mL propidium iodide (PI) for 5 min, and observed under a confocal microscope (Leica Micosystems, Wetzlar, Germany).

Preparation of IEL and MLNL

IEL were isolated using a modification of a previously published method 29. Briefly, reversed small intestine was incubated at 37°C with shaking at 150 rpm for 45 min with 5% FBS-HBSS. The supernatant was filtered through a glass wool mesh and centrifuged at 1,200 rpm for 10 min at room temperature. The acquired cell suspension was centrifuged through a 44%/70% discontinuous Percoll (Amersham Biosciences AB, Uppsala, Sweden) gradient at 1,800 rpm for 18 min. IEL were obtained from the interface of a discontinuous Percoll gradient.

To obtain MLNL, the mesenteric lymph nodes were surgically removed, crushed on a nylon mesh filter, and washed twice with 5% FBS-HBSS.

Flow cytometric analyses of IEL and MLNL

The IEL and MLNL obtained from individual mice of the same experimental group were pooled before flow cytometric analysis. Biotinylated anti-CD4 and phycoerythrin (PE)-conjugated anti-CD8a Ab (BD PharMingen, Tokyo, Japan) were used as the primary Ab and the avidin-conjugated fluorochrome Alexa 647 (BD PharMingen) was added as the secondary staining reagent for the biotinylated Ab. The GFP signal was also used as a marker of grafted donor cells. Flow cytometric analysis was performed using the EPICS ELITE ESP cell sorter (Beckman Coulter, Tokyo, Japan) with the EXPO32 software.

Quantitative RT-PCR for cytokine mRNA detection

The level of cytokine expression was examined by quantitative RT-PCR 43. The extracted RNA from IEL and MLNL was purified (RNeasy MinElute Cleanup Kit, Qiagen, Tokyo, Japan). and reverse-transcribed and amplified by PCR (ImProm-II Reverse Transcription System, Promega). Amplification was performed using the ABI PRISM 7000 Quantitative PCR System (Applied Biosystems, Tokyo, Japan). Each sample was examined in triplicate and the PCR products were normalized in relation to the hypoxanthine phosphoribosyltransferase (HPRT) internal control. The following primer sets were used: IL-4 (sense), 5′-CGCCATGCACGGAGATG-3′ and IL-4 (antisense), 5′-CGAGCTCACTCTCTGTGGTGTT-3′; IL-12p40 (sense), 5′-AGACCCTGCCCATTGAACTG-3′ and IL-12p40 (antisense), 5′-GAAGCTGGTGCTGTAGTTCTCATATT-3′; IL-18 (sense), 5′-AAGAAAGCCGCCTCAAACCT-3′ and IL-18 (antisense), 5′-TCTGACATGGCAGCCATTGT-3′; TNF-α (sense), 5′-GCTGTCGCTACATCATCGAACCT-3′ and TNF-α (antisense), 5′-TGACCCGTAGGGCGATTACA-3′; HPRT (sense), 5′-GCTCGAGATGTCATGAAGGAGAT-3′ and HPRT (antisense), 5′-AAAGAACTTATAGCCCCCCTTGA-3′.

In vitro viability and cell death assays of isolated IEL from GVHD mice

Isolated IEL from GVHD-induced mice were cultured in RPMI 1640 medium (Sigma-Aldrich, Tokyo, Japan) that contained 10% FBS and antibiotics. The IEL suspensions were counted and 5 × 105 cells were incubated in 1 mL of medium. A portion of the lymphocyte sample was stimulated by pretreatment with purified anti-TCR-β Ab (0.01 mg/mL for 60 min at 37°C; BD PharMingen) and soluble anti-CD28 Ab (0.5 μg/mL; BD PharMingen). The number of live cells was determined by trypan blue staining after 48 h of incubation with indicated medium. The supernatants were collected and assayed for cell death using the LDH release assay (CytoTox 96 Assay Kit; Promega, Tokyo, Japan) 45.

Estimation of TNF-α levels in bowel tissues

The ELISA assay for TNF-α (R&D Systems, Minneapolis, MN, USA) in gut homogenates was performed as described previously 25. Briefly, the bowel tissue was harvested and homogenized with a tissue homogenizer in nine volumes of lysis buffer. The cells were lysed by incubating on ice for 30 min, followed by two centrifugations (10 min, 14,000 × g, 4°C). The homogenates were stored at −20°C until use.

Statistical analysis

The values are presented as mean ± SEM per group. The results were analyzed using the two-tailed Student's t-test. The survival data were plotted by the Kaplan-Meier method and analyzed using the log-rank test. p values < 0.05 were considered statistically significant.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 7

    WILEY-VCH

  • 8

    WILEY-VCH

  • 1
    McDonald, G. B., Shulman, H. M., Sullivan, K. M. and Spencer, G. D., Intestinal and hepatic complications of human bone marrow transplantation. Part I. Gastroenterology 1986. 90: 460477.
  • 2
    McDonald, G. B., Shulman, H. M., Sullivan, K. M. and Spencer, G. D., Intestinal and hepatic complications of human bone marrow transplantation. Part II. Gastroenterology 1986. 90: 770784.
  • 3
    Shidham, V. B., Chang, C. C., Shidham, G., Ghazala, F., Lindholm, P. F., Kampalath, B., George, V. and Komorowski, R., Colon biopsies for evaluation of acute graft-versus-host disease (A-GVHD) in allogeneic bone marrow transplant patients. BMC Gastroenterol. 2003. 3: 5.
  • 4
    Hill, G. R., Crawford, J. M., Cooke, K. R., Brinson, Y. S., Pan, L. and Ferrara, J. L., Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood 1997. 90: 32043213.
  • 5
    Cooke, K. R., Hill, G. R., Crawford, J. M., Bungard, D., Brinson, Y. S., Delmonte, J., Jr. and Ferrara, J. L., Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J. Clin. Invest. 1998. 102: 18821891.
  • 6
    Brown, G. R., Lindberg, G., Meddings, J., Silva, M., Beutler, B. and Thiele, D., Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease. Gastroenterology 1999. 116: 593601.
  • 7
    Thiele, D. L., Eigenbrodt, M. L., Bryde, S. E., Eigenbrodt, E. H. and Lipsky, P. E., Intestinal graft-versus-host disease is initiated by donor T cells distinct from classic cytotoxic T lymphocytes. J. Clin. Invest. 1989. 84: 19471956.
  • 8
    Moriguchi, T., Kuroyanagi, N., Yamaguchi, K., Gotoh, Y., Irie, K., Kano, T., Shirakabe, K. et al., A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J. Biol. Chem. 1996. 271: 1367513679.
  • 9
    Han, J., Lee, J. D., Jiang, Y., Li, Z., Feng, L. and Ulevitch, R. J., Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J. Biol. Chem. 1996. 271: 28862891.
  • 10
    Jiang, Y., Gram, H., Zhao, M., New, L., Gu, J., Feng, L., Di Padova, F. et al., Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J. Biol. Chem. 1997. 272: 3012230128.
  • 11
    Li, Z., Jiang, Y., Ulevitch, R. J. and Han, J., The primary structure of p38 gamma: a new member of p38 group of MAP kinases. Biochem. Biophys. Res. Commun. 1996. 228: 334340.
  • 12
    Vachon, P. H., Harnois, C., Grenier, A., Dufour, G., Bouchard, V., Han, J., Landry, J. et al., Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis. Gastroenterology 2002. 123: 19801991.
  • 13
    Tamura, K., Sudo, T., Senftleben, U., Dadak, A. M., Johnson, R. and Karin, M., Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 2000. 102: 221231.
  • 14
    Wu, C. C., Hsu, S. C., Shih, H. M. and Lai, M. Z., Nuclear factor of activated T cells c is a target of p38 mitogen-activated protein kinase in T cells. Mol. Cell Biol. 2003. 23: 64426454.
  • 15
    Rincon, M., Enslen, H., Raingeaud, J., Recht, M., Zapton, T., Su, M. S., Penix, L. A. et al., Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 1998. 17: 28172829.
  • 16
    Lu, H. T., Yang, D. D., Wysk, M., Gatti, E., Mellman, I., Davis, R. J. and Flavell, R. A., Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J. 1999. 18: 18451857.
  • 17
    Schafer, P. H., Wadsworth, S. A., Wang, L. and Siekierka, J. J., p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J. Immunol. 1999. 162: 71107119.
  • 18
    Geginat, J., Sallusto, F. and Lanzavecchia, A., Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. J. Exp. Med. 2001. 194: 17111719.
  • 19
    Crawley, J. B., Rawlinson, L., Lali, F. V., Page, T. H., Saklatvala, J. and Foxwell, B. M., T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation. J. Biol. Chem. 1997. 272: 1502315027.
  • 20
    Ward, S. G., Parry, R. V., Matthews, J. and O'Neill, L., A p38 MAP kinase inhibitor SB203580 inhibits CD28-dependent T cell proliferation and IL-2 production. Biochem. Soc. Trans. 1997. 25: 304S.
  • 21
    Merritt, C., Enslen, H., Diehl, N., Conze, D., Davis, R. J. and Rincon, M., Activation of p38 mitogen-activated protein kinase in vivo selectively induces apoptosis of CD8(+) but not CD4(+) T cells. Mol. Cell Biol. 2000. 20: 936946.
  • 22
    Badger, A. M., Bradbeer, J. N., Votta, B., Lee, J. C., Adams, J. L. and Griswold, D. E., Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J. Pharmacol. Exp. Ther. 1996. 279: 14531461.
  • 23
    Jackson, J. R., Bolognese, B., Hillegass, L., Kassis, S., Adams, J., Griswold, D. E. and Winkler, J. D., Pharmacological effects of SB 220025, a selective inhibitor of p38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models. J. Pharmacol. Exp. Ther. 1998. 284: 687692.
  • 24
    Nick, J. A., Young, S. K., Brown, K. K., Avdi, N. J., Arndt, P. G., Suratt, B. T., Janes, M. S. et al., Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J. Immunol. 2000. 164: 21512159.
  • 25
    ten Hove, T., van den Blink, B., Pronk, I., Drillenburg, P., Peppelenbosch, M. P. and van Deventer, S. J., Dichotomal role of inhibition of p38 MAPK with SB 203580 in experimental colitis. Gut 2002. 50: 507512.
  • 26
    Ellison, C. A., Fischer, J. M., HayGlass, K. T. and Gartner, J. G., Murine graft-versus-host disease in an F1-hybrid model using IFN-gamma gene knockout donors. J. Immunol. 1998. 161: 631640.
  • 27
    Takanami-Ohnishi, Y., Amano, S., Kimura, S., Asada, S., Utani, A., Maruyama, M. et al., Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity. J. Biol. Chem. 2002. 277: 3789637903.
  • 28
    Bombi, J. A., Nadal, A., Carreras, E., Ramirez, J., Munoz, J., Rozman, C. and Cardesa, A., Assessment of histopathologic changes in the colonic biopsy in acute graft-versus-host disease. Am. J. Clin. Pathol. 1995. 103: 690695.
  • 29
    Sakai, T., Kimura, Y., Inagaki-Ohara, K., Kusugami, K., Lynch, D. H. and Yoshikai, Y., Fas-mediated cytotoxicity by intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice. Gastroenterology 1997. 113: 168174.
  • 30
    Antin, J. H. and Ferrara, J. L., Cytokine dysregulation and acute graft-versus-host disease. Blood 1992. 80: 29642968.
  • 31
    Visconti, R., Gadina, M., Chiariello, M., Chen, E. H., Stancato, L. F., Gutkind, J. S. and O'Shea, J. J., Importance of the MKK6/p38 pathway for interleukin-12-induced STAT4 serine phosphorylation and transcriptional activity. Blood 2000. 96: 18441852.
  • 32
    Fuss, I. J., Marth, T., Neurath, M. F., Pearlstein, G. R., Jain, A. and Strober, W., Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice. Gastroenterology 1999. 117: 10781088.
  • 33
    Marth, T., Strober, W. and Kelsall, B. L., High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-beta secretion and T cell apoptosis. J. Immunol. 1996. 157: 23482357.
  • 34
    Estaquier, J., Idziorek, T., Zou, W., Emilie, D., Farber, C. M., Bourez, J. M. and Ameisen, J. C., T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. J. Exp. Med. 1995. 182: 17591767.
  • 35
    Radrizzani, M., Accornero, P., Amidei, A., Aiello, A., Delia, D., Kurrle, R. and Colombo, M. P., IL-12 inhibits apoptosis induced in a human Th1 clone by gp120/CD4 cross-linking and CD3/TCR activation or by IL-2 deprivation. Cell Immunol. 1995. 161: 1421.
  • 36
    Brown, G. R. and Thiele, D. L., T-cell activation and differentiation are regulated by TNF during murine DBA/2-->B6D2F1 intestinal graft-versus-host disease. J. Clin. Immunol. 2000. 20: 379388.
  • 37
    Hill, G. R., Teshima, T., Rebel, V. I., Krijanovski, O. I., Cooke, K. R., Brinson, Y. S. and Ferrara, J. L., The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity. J. Immunol. 2000. 164: 656663.
  • 38
    Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D. and Gaestel, M., MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat. Cell Biol. 1999. 1: 9497.
  • 39
    van den Blink, B., Juffermans, N. P., ten Hove, T., Schultz, M. J., van Deventer, S. J., van der Poll, T. and Peppelenbosch, M. P., p38 mitogen-activated protein kinase inhibition increases cytokine release by macrophages in vitro and during infection in vivo. J. Immunol. 2001. 166: 582587.
  • 40
    Schafer, P. H., Wang, L., Wadsworth, S. A., Davis, J. E. and Siekierka, J. J., T cell activation signals up-regulate p38 mitogen-activated protein kinase activity and induce TNF-alpha production in a manner distinct from LPS activation of monocytes. J. Immunol. 1999. 162: 659668.
  • 41
    Doyle, S. E., O'Connell, R. M., Miranda, G. A., Vaidya, S. A., Chow, E. K., Liu, P. T., Suzuki, S. et al., Toll-like receptors induce a phagocytic gene program through p38. J. Exp. Med. 2004. 199: 8190.
  • 42
    Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y., ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 1997. 407: 313319.
  • 43
    Kuroiwa, T., Kakishita, E., Hamano, T., Kataoka, Y., Seto, Y., Iwata, N., Kaneda, Y. et al., Hepatocyte growth factor ameliorates acute graft-versus-host disease and promotes hematopoietic function. J. Clin. Invest. 2001. 107: 13651373.
  • 44
    Tanaka, K., Sata, M., Hirata, Y. and Nagai, R., Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ. Res. 2003. 93: 783790.
  • 45
    Brander, C., Wyss-Coray, T., Mauri, D., Bettens, F. and Pichler, W. J., Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide. Eur. J. Immunol. 1993. 23: 32173223.