Serial transplantation of p53-deficient hemopoietic progenitor cells to assess their infinite growth potential

Thirty-five years ago, Siminovitch et al. (Siminovitch L, Till JE, McCulloch EA. J Cell Com Physiol 64:23-32, 1964), using serially transplanted mouse spleens at 14-day intervals, observed a markedly progressive decline in the proliferative capacity of bone marrow (BM) cells, with the loss of clonogenicity by the fourth transplant generation. Using the same protocol, we assessed the proliferative capacity of p53-deficient mouse BM cells transplanted serially at the same 14-day intervals into lethally irradiated mice, which was a useful tool for understanding the characteristics of hemopoietic stem cells lacking solely the p53 gene function. BM cells from p53-deficient homozygous (p53(-/-)), p53-heterozygous (p53(+/-)), and wild-type (p53(+/+)) C57BL/6 mice were transplanted into lethally irradiated C57BL/6 recipients. Fourteen days later, the repopulated spleens were harvested, and 10(7)cells were retransplanted into secondary recipients. Serial transplantation was continued at 14-day intervals until hemopoietic repopulation failure. The number of heterozygous and homozygous p53-deficient spleen cells increased logarithmically up to the fourth and fifth passages, respectively, whereas wild-type spleen cells ceased to proliferate by the third passage. The number of macroscopic spleen colonies increased logarithmically until the third passage in recipients of heterozygous and homozygous p53-deficient cells, but ceased to grow by the second passage in recipients of wild-type cells. The numbers of heterozygous and homozygous p53-deficient colony forming units in spleen (CFUs-S) remained stable during the first four transplant generations, whereas that of wild-type CFUs-S decreased progressively from the first transplant generation onward. The clonogenicity of p53-deficient cells was lost when the number of CFUs-S per spleen decreased to below 10. This suggests that one out of 10 CFUs-S might be long-term repopulating cells (LTRCs), and that p53-deficient LTRCs may proliferate more rapidly than wild-type LTRCs. Longer passages that were possible in the p53-deficient groups were considered to be due to the faster cell cycle of the p53-deficient hemopoietic progenitor cells, as determined by bromodeoxyuridine incorporation with purging by UV light exposure, followed by hemopoietic colony assay (BUUV assay).     

Bone-marrow chimeras reveal hemopoietic and nonhemopoietic control of resistance to experimental Lyme arthritis

Both genetic resistance and susceptibility to development of experimental Lyme arthritis are mediated by the innate immune response. To determine whether this process is mainly controlled by hemopoietic or nonhemopoietic cells, we created bone marrow (BM) chimeric mice between arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice and infected them with Borrelia burgdorferi. Both sets of BM chimeric mice, C3H donors into DBA recipients (C-->D) and DBA donors into C3H recipients (D-->C), as well as DBA sham chimeric mice (D-->D) were resistant to the development of experimental Lyme arthritis as measured by ankle swelling and arthritis severity scores. Only the C3H sham chimeric mice (C-->C) developed severe arthritis. These results indicate that independent and nonoverlapping mechanisms exist in hemopoietic and nonhemopoietic cellular compartments that can provide protection against arthritic pathology.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

IL-18, but not IL-12, regulates NK cell activity following intranasal herpes simplex virus type 1 infection

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18(-/-)) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12(-/-)) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18(-/-) mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-gamma or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18(-/-) mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12(-/-) mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18(-/-) mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.     

Genetic differences in bone marrow-derived lymphoid lineages control susceptibility to experimental autoimmune myocarditis

Bone marrow (BM) transplantation has been used to study the cellular basis of genetic control of autoimmune diseases, but conclusions remain elusive due to the contradictory findings in different animal models. In the current study, we found that BM cells from myocarditis-susceptible A.SW mice can render irradiated, myocarditis-resistant B10.S recipient mice susceptible to myosin-induced myocarditis, indicating that hematopoietic cells express the genetic differences controlling susceptibility to autoimmune myocarditis. We then sought to differentiate the role of lymphoid vs nonlymphoid components of BM in the pathogenesis of myocarditis by comparing mixed chimeras receiving BM from A.SW wild-type or RAG(-/-) mice mixed with BM from B10.S wild-type mice. This experiment clearly demonstrated that T and B lymphocytes were indispensable for transferring the susceptible phenotype to disease-resistant recipients. Our findings significantly narrow the cellular expression of genetic polymorphisms controlling the EAM phenotype.     

Strain-specific effects of riboflavin supplementation on zymosan-induced peritonitis in C57BL/6J, BALB/c and CBA mice

AimsWe investigated the effects of riboflavin (vitamin B2) on the kinetics of zymosan-induced peritonitis in three strains of mice.Main methodsPeritonitis was induced in males of C57BL/6J, BALB/c and CBA mice by intraperitoneal injection of zymosan (40 mg/kg) or zymosan supplemented with riboflavin (50 mg/kg). During the first 45 min of inflammation the pain symptoms were scored. At the selected time points (4, 6, 8, 10, 24, and 30 h) the mice were sacrificed and peritoneal exudates were retrieved. Leukocytes, among them polymorphonuclear cells (PMNs) and macrophages (Mac3⁺ cells) were counted. Levels of inducible nitric oxide synthase (iNOS) were measured in cell pellets while supernatants were used for measurements of nitric oxide, cytokine/chemokines (IL-6, IL-10, MCP-1, IFNγ, TNF-α, and IL-12p70), and matrix metalloproteinase-9 (MMP-9).Key findingA riboflavin ip injection induced pain symptoms itself, but reduced zymosan-induced pain in C57BL/6J and CBA strains of mice when coinjected with zymosan. In comparison with the mice injected with zymosan only, riboflavin coinjection prolonged inflammation in C57BL/6J mice due to prolonged macrophage accumulation; inhibited peritoneal leukocytes (PTL) accumulation in BALB/c due to inhibited influx of macrophages and PMNs; and inhibited PTL accumulation in CBA mice due to delayed PMN influx. These effects corresponded with the delayed (C57BL/6J) or inhibited (BALB/c and CBA) expression of iNOS in PTL lysates, and with the prolonged (C57BL/6) or inhibited (BALB/c) intraperitoneal accumulation of MMP-9. Moreover, cytokine accumulation was affected in a strain-specific way.SignificanceRiboflavin is antinociceptive during yeast-induced peritonitis, but its anti-inflammatory effects are strain-specific.     

IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10-/-) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10-/- mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10-/- mice with either anti-CD4 or anti-IFN-gamma mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-gamma mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10-/- mice than those from infected control mice. In common with C57BL/6-background IL-10-/- mice, BALB/c-background IL-10-/- mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-gamma mAb prevented the pathology and prolonged time to death of the infected IL-10-/- mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-gamma production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-gamma-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.     

Staphylococcal enterotoxin B stimulates expansion of autoreactive T cells that induce apoptosis in intestinal epithelial cells: regulation of autoreactive responses by IL-10

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vbeta6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.     

IL-10 modulates intestinal damage and epithelial cell apoptosis in T cell-mediated enteropathy

In vivo T cell activation by anti-CD3 monoclonal antibody (mAb) results in intestinal damage characterized by loss of villi and epithelial cell apoptosis. The role of the increased interleukin (IL)-10 released during this process is not clear. We assessed the effects of IL-10 on T cell-induced mucosal damage in vivo using IL-10-deficient C57BL/6 [IL-10 knockout (KO)] mice. IL-10 KO and wild-type C57BL/6 mice were injected with anti-CD3 mAb and observed for diarrhea. Changes in serum cytokine levels were measured by ELISA. Histological changes and epithelial cell apoptosis were analyzed on hematoxylin- and eosin-stained tissue sections. Fas expression on intestinal epithelial cells was assessed by flow cytometry analysis of freshly isolated intestinal epithelial cells. Anti-CD3-treated IL-10 KO mice developed more severe diarrhea, a greater loss of intestinal villi, and an increase in the numbers of apoptotic cells in the crypt epithelium. This difference in IL-10 KO mice was associated with an increase in serum tumor necrosis factor-alpha and interferon-gamma levels and with an increase in Fas expression on fresh, isolated, small intestinal epithelial cells. In addition, the enhanced intestinal tissue damage induced by anti-CD3 in IL-10 KO mice was significantly diminished by treatment with recombinant murine IL-10. Therefore, the lack of IL-10 allowed for an increased T cell-induced intestinal tissue damage, and this was associated with an increase in T cell cytokine release and an increase in epithelial cell Fas expression.     

Resistance to metastatic disease in STAT6-deficient mice requires hemopoietic and nonhemopoietic cells and is IFN-gamma dependent

Mice deficient for the STAT6 gene (STAT6(-/-) mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8(+) T cells. Immunity in STAT6(-/-) mice is unusually effective in that 45-80% of STAT6(-/-) mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT(+/+) (BALB/c) mice. Surprisingly, STAT6(-/-) and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6(-/-) hemopoietic and nonhemopoietic components. Likewise, CD1(-/-) mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6(-/-) and CD1(-/-) reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6(-/-) and CD1(-/-) mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4(+)CD25(+) T cells in BALB/c mice does not increase survival, demonstrating that CD4(+)CD25(+) cells do not regulate immunity. Cytokine production and tumor challenges into STAT6(-/-)IFN-gamma(-/-) mice indicate that IFN-gamma is essential for immunity. Therefore, immunosurveillance in STAT6(-/-) mice facilitates survival against metastatic cancer via an IFN-gamma-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4(+)CD25(+) T cells.     

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.     

T Cell-Derived IL-10 Determines Leishmaniasis Disease Outcome and Is Suppressed by a Dendritic Cell Based Vaccine

In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.     

Responses of inbred mouse strains to infection with intestinal nematodes

Comparisons were made of the immune and inflammatory responses of four strains of inbred mice to infection with the intestinal nematodes Trichinella spiralis and Nippostrongylus brasiliensis to determine whether genetically determined 'high responsiveness' to infection, seen most clearly in intestinal responses, is independent of the parasite concerned and necessarily correlated with protection. The time course of infection was followed by counting adult worms at intervals after infection. Mucosal mast cells and Paneth cell numbers were determined as indices of the intestinal inflammatory response. Levels of IgG2a and IgG1 antibodies and of the cytokines IFN-gamma and IL-5 released from in vitro-stimulated mesenteric node lymphocytes were measured to assess type 1 and type 2 responses. NIH and CBA mice were the most resistant to T. spiralis and N. brasiliensis respectively, resistance in each case being correlated with the most intense intestinal inflammatory responses. C57BL/10 (B10) and B10.BR were the least resistant to T. spiralis, but were as resistant as CBA to N. brasiliensis, despite their intestinal inflammatory responses to both parasites being much lower than the other two strains. Mice infected with T. spiralis made the expected switch from a type 1 (IFN-gamma) to a type 2 (IL-5) response between days 2 and 8, and there were no significant differences in levels of these cytokines between the strains. In contrast, when infected with N. brasiliensis, CBA showed an IFN-gamma response at day 4, all strains switching to IL-5 by day 8 and NIH mice releasing the greatest amount of IL-5. The results indicate that the "high responder" phenotype to intestinal nematode infection is in part determined by host characteristics, but is also determined by the parasite concerned--seen most clearly by the differences between NIH and CBA when infected with T. spiralis and N. brasiliensis. The fact that "low responder" B10 background mice were more resistant to N. brasiliensis than "high responder" NIH implies that each parasite elicits a particular pattern of protective host responses, rather than parasites being differentially susceptible to the same response profile.     

MyD88-dependent signaling for IL-15 production plays an important role in maintenance of CD8 alpha alpha TCR alpha beta and TCR gamma delta intestinal intraepithelial lymphocytes

Interaction between commensal bacteria and intestinal epithelial cells (i-ECs) via TLRs is important for intestinal homeostasis. In this study, we found that the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta intestinal intraepithelial lymphocytes (i-IELs) were significantly decreased in MyD88-deficient (-/-) mice. The expression of IL-15 by i-ECs was severely reduced in MyD88(-/-) mice. Introduction of IL-15 transgene into MyD88(-/-) mice (MyD88(-/-) IL-15 transgenic mice) partly restored the numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs. The i-IEL in irradiated wild-type (WT) mice transferred with MyD88(-/-) bone marrow (BM) cells had the same proportions of i-IEL as WT mice, whereas those in irradiated MyD88(-/-) mice transferred with WT BM cells showed significantly reduced proportions of CD8alphaalpha TCRalphabeta and TCRgammadelta i-IELs, as was similar to the proportions found in MyD88(-/-) mice. However, irradiated MyD88(-/-) IL-15 transgenic mice transferred with WT BM cells had increased numbers of CD8alphaalpha TCRalphabeta and TCRgammadelta subsets in the i-IEL. These results suggest that parenchymal cells such as i-ECs contribute to the maintenance of CD8alphaalpha TCRalphabeta and gammadelta i-IELs at least partly via MyD88-dependent IL-15 production.     

The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice

Gastritis due to Helicobacter pylori in mice and humans is considered a Th1-mediated disease, but the specific cell subsets and cytokines involved are still not well understood. The goal of this study was to investigate the immunopathogenesis of H. pylori-induced gastritis and delayed-type hypersensitivity (DTH) in mice. C57BL/6-Prkdc(scid) mice were infected with H. pylori and reconstituted with CD4+, CD4-depleted, CD4+CD45RB(high), or CD4+CD45RB(low) splenocytes from wild-type C57BL/6 mice or with splenocytes from C57BL/6(IFN-gamma-/-) or C57BL/6(IL-10-/-) mice. Four or eight weeks after transfer, DTH to H. pylori Ags was determined by footpad injection; gastritis and bacterial colonization were quantified; and IFN-gamma secretion by splenocytes in response to H. pylori Ag was determined. Gastritis and DTH were present in recipients of unfractionated splenocytes, CD4+ splenocytes, and CD4+CD45RB(high) splenocytes, but absent in the other groups. IFN-gamma secretion in response to H. pylori Ags was correlated with gastritis, although splenocytes from all groups of mice secreted some IFN-gamma. Gastritis was most severe in recipients of splenocytes from IL-10-deficient mice, and least severe in those given IFN-gamma-deficient splenocytes. Bacterial colonization in all groups was inversely correlated with gastritis. These data indicate that 1) CD4+ T cells are both necessary and sufficient for gastritis and DTH due to H. pylori in mice; 2) high expression of CD45RB is a marker for gastritis-inducing CD4+ cells; and 3) IFN-gamma contributes to gastritis and IL-10 suppresses it, but IFN-gamma secretion alone is not sufficient to induce gastritis. The results support the assertion that H. pylori is mediated by a Th1-biased cellular immune response.     

Distinct subsets of dendritic cells regulate the pattern of acute xenograft rejection and susceptibility to cyclosporine therapy

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c(+)CD8alpha(+) splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c(+)CD8alpha(-) splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c(+)IL-12(+) splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8alpha(-) bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8alpha(+) bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 +/- 0.6 days to 14.2 +/- 0.8 days. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.     

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.     

IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12

IL-23, a heterodimeric cytokine composed of the p40 subunit of IL-12 and a novel p19 subunit, has been shown to be a key player in models of autoimmune chronic inflammation. To investigate the role of IL-23 in host resistance during chronic fungal infection, wild-type, IL-12- (IL-12p35-/-), IL-23- (IL-23p19-/-), and IL-12/IL-23- (p40-deficient) deficient mice on a C57BL/6 background were infected with Cryptococcus neoformans. Following infection, p40-deficient mice demonstrated higher mortality than IL-12p35-/- mice. Reconstitution of p40-deficient mice with rIL-23 prolonged their survival to levels similar to IL-12p35-/- mice. IL-23p19-/- mice showed a moderately reduced survival time and delayed fungal clearance in the liver. Although IFN-gamma production was similar in wild-type and IL-23p19-/- mice, production of IL-17 was strongly impaired in the latter. IL-23p19-/- mice produced fewer hepatic granulomata relative to organ burden and showed defective recruitment of mononuclear cells to the brain. Moreover, activation of microglia cells and expression of IL-1beta, IL-6, and MCP-1 in the brain was impaired. These results show that IL-23 complements the more dominant role of IL-12 in protection against a chronic fungal infection by an enhanced inflammatory cell response and distinct cytokine regulation.