Intrinsic hyporesponsiveness of invariant natural killer T cells precedes the onset of lupus.

Patients with systemic lupus erythematosus (SLE) display reduced numbers and functions of invariant natural killer T (iNK T) cells, which are restored upon treatment with corticosteroids and rituximab. It is unclear whether the iNK T cell insufficiency is a consequence of disease or is a primary abnormality that precedes the onset of disease. To address this, we analysed iNK T cell function at different stages of disease development using the genetically lupus-susceptible NZB × NZW F1 (BWF(1)) model. We found that iNK T cell in-vivo cytokine responses to an iNK T cell ligand α-galactosylceramide (α-GalCer) were lower in BWF(1) mice than in non-autoimmune BALB/c and major histocompatibility complex (MHC)-matched NZB × N/B10.PL F1 mice, although iNK T cell numbers in the periphery were unchanged in BWF(1) mice compared to control mice. Such iNK T cell hyporesponsiveness in BWF(1) mice was detected at a young age long before the animals exhibited any sign of autoimmunity. In-vivo activation of iNK T cells is known to transactivate other immune cells. Such transactivated T and B cell activation markers and/or cytokine responses were also lower in BWF(1) mice than in BALB/c controls. Finally, we show that iNK T cell responses were markedly deficient in the NZB parent but not in NZW parent of BWF(1) mice, suggesting that BWF(1) might inherit the iNK T cell defect from NZB mice. Thus, iNK T cells are functionally insufficient in lupus-prone BWF(1) mice. Such iNK T cell insufficiency precedes the onset of disease and may play a pathogenic role during early stages of disease development in SLE.

Resistance to malarial infection is achieved by the cooperation of NK1.1(+) and NK1.1(-) subsets of intermediate TCR cells which are constituents of innate immunity.

We previously reported that the major expanding lymphocytes were intermediate TCR (TCR(int)) cells (mainly NK1.1(-)) during malarial infection in mice. Cell transfer experiments of TCR(int) cells indicated that these T cells mediated resistance to malaria. However, TCR(int) cells always contain NK1.1(+)TCR(int) cells (i.e., NKT cells) and controversial results (NKT cells were effective or not for resistance to malaria) have been reported by different investigators. In this study, we used CD1d((-/-)) mice, which almost completely lack NKT cells in the liver and other immune organs. Parasitemia was prolonged in the blood of CD1d((-/-)) mice and the expansion of lymphocytes in the liver of these mice was more prominent after an injection of Plasmodium yoelii-infected erythrocytes. However, these mice finally recovered from malaria. In contrast to B6 mice, CD4(-)8(-) NKT cells as well as NK1.1(-)CD3(int) cells expanded in CD1d((-/-)) mice after malarial infection, instead of CD4(+) (and CD8(+)) NKT cells. These newly generated CD4(-)8(-)NKT cells in CD1d((-/-)) mice did not use an invariant chain of Valpha14Jalpha281 for TCRalpha. Other evidence was that severe thymic atrophy and autoantibody production were accompanied by malarial infection, irrespective of the mice used. These results suggest that both NK1.1(-) and NK1.1(+) subsets of TCR(int) cells (i.e., constituents of innate immunity) are associated with resistance to malaria and that an autoimmune-like state is induced during malarial infection.

Intensive generation of NK1.1- extrathymic T cells in the liver by injection of bone marrow cells isolated from mice with a mutation of polymorphic major histocompatibility complex antigens.

Whether intermediate TCR (TCRint) cells and natural killer T (NKT or NK1.1+TCRint) cells are extrathymically generated remains controversial. This arises from the fact that there are few of these T cells in athymic nude mice and neonatally thymectomized mice. However, when athymic mice were provided with appropriate microenvironments or stimulation, many TCRint cells (mainly NK1.1-) were found to arise in the liver. NKT cells are known to be positively selected by monomorphic major histocompatibility complex (MHC) -like antigens (e.g. CD1d). This is true even if they are CD4+. In other words, a MHC class I-like antigen is restricted to CD4 antigen. This rule is somewhat different from that seen in conventional T cells (i.e. the restriction of class II with CD4 and that of class I and CD8). In the case of NK1.1-TCRint cells, they were selected by polymorphic MHC antigens, but their MHC restriction to CD4 or CD8 antigen was incomplete. This was revealed by experiments of bone marrow transfer with class I (bm 1) or II (bm 12) disparity. Depending on the disparity, a unique cytokine profile in sera was detected. These results suggest that the development of T lineage lymphocytes and MHC restriction to CD4 and CD8 might have occurred in parallell as a phylogenic event, and that NK1.1- extrathymic T cells (i.e. NK1.1-TCRint) are at an intermediate position between NKT cells and conventional T cells in phylogeny.

Mechanisms involved in enteropathy induced by administration of nonsteroidal antiinflammatory drugs (NSAIDS).

Mice received oral indomethacin (1 mg/mouse) daily for five days. It was found that severe gastroenteropathy (ie, paralytic stomach and necrotic intestine) was induced on the sixth day. Ulcer formation was also seen at many sites in the digestive tract, especially in the colon. In parallel with the increase in the number of leukocytes in the digestive tract, the proportion of granulocytes increased at various sites, for example, in the intraepithelium and lamina propria of the colon and the lamina propria of the appendix. The number of extrathymic T cells at these sites in the digestive tract, especially gammadelta T cells in the colon, increased. A functional assay revealed that granulocytes isolated from mice injected with indomethacin were activated in terms of their superoxide production upon stimulation. In conjunction with the data on the simultaneous activation of granulocytes in the liver and blood, the present results suggest that nonsteroidal antiinflammatory drugs (NSAIDs) have the potential to induce severe granulocytosis in specific sites of the body, possibly via their stimulatory effect on the sympathetic nervous system (ie, granulocytes bear adrenergic receptors on their surface).

Association of intermediate T cell receptor cells, mainly their NK1.1(-) subset, with protection from malaria.

Mice were infected with Plasmodium (P.) yoelii blood-stage parasites. Both the liver and spleen were the sites of inflammation during malarial infection at the beginning of day 7. The major expanding cells were found to be NK1.1(-) intermediate alphabetaTCR (alphabetaTCR(int)) in the liver and spleen, although the population of NK1.1(+) alphabetaTCR(int) cells remained constant or slightly increased. These TCR(int) cells are of extrathymic origin or are generated by an alternative intrathymic pathway and are distinguished from conventional T cells of thymic origin. During malarial infection, the population of conventional T cells did not increase at all. TCR(int) cells purified from the liver of mice which had recovered from P. yoelii infection protected mice from malaria when they were transferred into 6.5-Gy-irradiated mice. Interestingly, the immunity against malaria seemed to disappear as a function of time after recovery, namely, mice which had recovered from malaria 1 year previously again became susceptible to malarial infection. The present results suggest that TCR(int) cells are intimately associated with protection against malarial infection and, therefore, that mice which had recovered from malaria 1 year previously lost such immunity.

Resistance of extrathymic T cells to stress and the role of endogenous glucocorticoids in stress associated immunosuppression.

When mice were exposed to restraint stress for 12 or 24 h, severe lymphopenia was induced in all immune system organs, including the liver and the thymus. However, in adrenalectomized mice, this response was completely absent. Phenotypic characterization revealed that interleukin (IL)-2Rbeta+CD3int cells (i.e. extrathymic T cells) with CD4+ phenotype and the NK1.1+ subset of CD3int cells (i.e. NKT cells) in the liver as well as the mature conventional T cells in the thymus were resistant to such stress. In adrenalectomized mice, there was no significant change in the distribution of lymphocyte subsets in all tested organs before stress. Interestingly, the number of lymphocytes in the liver and spleen and the proportion of NKT cells in the liver rather increased after stress in these adrenalectomized mice. Therefore, endogenous steroid hormones were indicated to be important in the induction of immunosuppressive states after stress. Among stress associated cytokines, the secretion of tumour necrosis factor (TNF)-alpha was completely suppressed while that of IL-6 was partially suppressed in adrenalectomized mice. These results suggest that endogenous steroid hormones are important for the induction of the stress associated immunosuppression and that NKT cells are resistant to stress, namely, resistant to exposure to endogenous steroid hormones.

Consistent infiltration of thymus-derived T cells into the parenchymal space of the liver in normal mice.

We previously reported that extrathymic T cells (intermediate T-cell receptor cells [TCR(int) cells]) are in situ generated in the parenchymal space of the liver in mice. They subsequently migrate to the sinusoidal lumen. In this study, we characterized how such extrathymic T cells, natural killer (NK) cells, and thymus-derived T cells (high T-cell receptor cells [TCR(high) cells]) localized in the parenchymal space or the sinusoidal lumen of mice. To this end, liver irrigation with physiological saline from the portal vein was performed and the distribution of lymphocyte subsets was compared between the liver (i.e., lymphocytes in the parenchymal space) and the irrigation solution (i.e., lymphocytes in the sinusoidal lumen). Extrathymic T cells and NK cells were found to be abundant in both the liver and sinusoidal lumen. As expected, thymus-derived T cells were abundant in the sinusoidal lumen. However, a significant proportion of thymus-derived T cells were always present in the parenchymal space, even after intensive irrigation with or without collagenase. These results suggest that thymus-derived T cells may consistently infiltrate the parenchymal space from the sinusoidal lumen in normal mice. This possibility was confirmed by (1) the injection of B6 splenic cells (TCR(high) cells) or the thymus graft into B6-nu/nu mice (presence of only TCR(int) cells) and by (2) using parabiotic mice of B6.Ly5.1 and B6.Ly5.2 strains (sharing circulation) in conjunction with immunofluorescence tests and immunohistochemical staining. In other words, inverted routes of migration and homing between extrathymic T cells and thymus-derived T cells exist in the liver.

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield.

We previously reported that c-kit+ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3- IL-2Rbeta+) and extrathymic T cells (CD3int IL-2Rbeta+) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8+ CD3int IL-2Rbeta+ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1+ and NK1.1- subsets of CD3int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1+ subset of CD3int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1- subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.

Lipopolysaccharide-specific antibodies in plasma and stools of children with Shigella-associated leukemoid reaction and hemolytic-uremic syndrome.

Antibody responses to the lipopolysaccharide (LPS) of shigellae were compared between children with uncomplicated and complicated Shigella dysenteriae 1 infection. One hundred fifteen children between 12 and 60 months of age with S. dysenteriae 1 infection were studied. Of these children, 42 had complications (leukemoid reaction and/or hemolytic-uremic syndrome [complicated shigellosis] and 73 had no complications (uncomplicated shigellosis). Antibodies to the LPS of S. dysenteriae 1 and Shigella flexneri Y were measured in plasma and stools, as were total immunoglobulin A (IgA) and IgG concentrations in plasma and the total IgA concentration in stool, on enrollment and 3 to 5 days later. In the plasma, the concentrations of homologous (IgG) and heterologous (IgA) LPS antibodies on enrollment were higher in children with complicated shigellosis than in those with uncomplicated shigellosis. In stool, the concentrations on enrollment were similar between the two groups of children. There was a rise in antibody concentrations in the plasma (homologous and heterologous) and stool (homologous) between the day of enrollment and 3 to 5 days later in children with uncomplicated shigellosis but not in those with complicated shigellosis. These findings suggest that systemic stimulation is more marked in children with complications, so that a subsequent rise in plasma antibody concentrations does not occur in these children. In contrast, the lack of a rise in stool antibody concentrations in children with complicated shigellosis is suggestive of a lower-level mucosal response. Because the duration of diarrhea before enrollment influenced the homologous antibody concentrations, children were further divided into three subgroups (short [3 to 5 days], medium [6 to 9 days], and long [> 9 days] diarrhea durations before enrollment). Comparisons of homologous antibody concentrations between the two groups of children following such subdivisions showed that in children with complicated shigellosis, antibody concentrations were higher in the plasma of children in the short diarrhea duration subgroup but lower in the stool children in the medium diarrhea duration subgroup. No differences in antibody concentrations were observed in children in the other diarrhea duration subgroups. Thus, complications in shigellosis are associated with an early and strong systemic stimulation without a concomitant stimulation of the mucosal antibody response.

Alterations in lymphocyte phenotype and function in children with shigellosis who develop complications.

This study was designed to see whether alterations occur in peripheral blood mononuclear cell phenotype and function in children with Shigella dysenteriae 1 infection with complications (leukemoid reaction and/or hemolytic-uremic syndrome) and whether there are any alterations prior to the development of complications. The following groups of children (ages, 12 to 60 months) were compared: children without any infection (n = 51), children with uncomplicated shigellosis (n = 65), children admitted with complicated shigellosis (leukemoid reaction and/or hemolytic-uremic syndrome) (n = 29), and children with shigellosis who developed complications after enrollment (subsequently complicated shigellosis) (n = 12). Tests for the peripheral blood mononuclear cell phenotype (CD3, CD4, CD8, CD57 [corrected], CD20, and CD25), spontaneous proliferation, and the proliferative response to phytohemagglutinin, pokeweed mitogen, and the lipopolysaccharide of S. dysenteriae 1 were performed, as were skin tests for delayed-type hypersensitivity (DTH). Children who subsequently developed complications differed from other groups of children as follows: (i) the numbers of CD3+ and CD4+ cells were lower than in uninfected children (P < 0.05), (ii) the CD4/CD8 ratio was lower than in children with uncomplicated shigellosis (P < 0.05) and in uninfected children (P < 0.05), and (iii) the levels of spontaneous proliferation of peripheral blood mononuclear cells were higher and DTH responses were lower than those in children with uncomplicated shigellosis (P < 0.05 and P < 0.017, respectively). Children with complications differed by having (i) increased numbers of CD3- CD57- [corrected] CD20- cells (P < 0.05) compared with those in other groups of children and (ii) lower CD4/CD8 ratios (P < 0.05), higher levels of spontaneous proliferation (P < 0.05), and lower DTH responses (P = 0.005) than children with uncomplicated shigellosis. Three to five days after enrollment, the number of CD4+ cells increased in children who subsequently developed complications (P = 0.025), i.e., when they developed complications and at this time their CD4+ cell number was similar to that of other groups of children. Thus, lymphocyte phenotype and function are altered prior to the development of complications in children with shigellosis, and once complications develop, the pattern of alterations changes. Whether these alterations have a role in precipitating complications or whether they reflect early events underlying the development of complications remains to be elucidated.

Peripheral blood neutrophil responses in children with shigellosis.

Alterations in peripheral blood neutrophil function are known to occur in patients with colitis and may have a role in precipitating nonspecific tissue injury. It is not known whether neutrophil function is altered in patients with Shigella dysenteriae type 1 infection, during which there is extensive colitis and which may be associated with life-threatening complications in young children. Three aspects of peripheral blood neutrophil function, polarization, attachment to yeast particles, and locomotion, were therefore studied in 111 children with S. dysenteriae type 1 infection and 57 children without any infection. All children were aged 12 to 60 months. Of the children with S. dysenteriae type 1 infection, 42 had leukemoid reaction, hemolytic-uremic syndrome, or septicemia (complicated shigellosis), while the others did not (uncomplicated shigellosis). Polarization and locomotion in the absence of chemoattractants and in response to N-formylmethionyl-leucylphenylalanine (FMLP) and the lipopolysaccharide (LPS) of S. dysenteriae type 1 were determined. Attachment to unopsonized and opsonized yeast particles was also determined. Children with shigellosis (uncomplicated or complicated) had more polarized neutrophils with and without chemoattractants than uninfected children (P < 0.05). Children with complicated shigellosis had more polarized neutrophils with FMLP at 10(-7) and 10(-6) M (P < 0.05) and with LPS than children with uncomplicated shigellosis (P < 0.05). At 3 to 5 days after enrollment, the numbers of polarized neutrophils with 10(-8), 10(-6), and 10(-5) M FMLP declined in children with uncomplicated shigellosis but not in those with complicated shigellosis. Attachment to yeast particles was similar in all three groups of children. Locomotion was inhibited by LPS in children with shigellosis (P < 0.05), whether it was uncomplicated or complicated, compared with locomotion in uninfected children. Finally, neutrophil polarization in uninfected children was negatively influenced by nutritional status. Thus, poorly nourished uninfected children had more polarized neutrophils with FMLP at 10(-9) M (P < = 0.02) and 10(-5) M (P = 0.043) than their better-nourished counterparts. In summary, altered neutrophil responses are associated with both uncomplicated and complicated shigellosis.

Cytokines in the stools of children with complicated shigellosis.

The pathogenesis of the systemic complications, leukemoid reaction and hemolytic uremic syndrome, associated with Shigella dysenteriae type 1 infection is not well understood. The excessive production of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), has been suggested as a possible factor. We measured IL-6 and TNF-alpha in stools of 56 children with S. dysenteriae 1 infection and 29 children without any apparent infection, all age 12 to 60 months. Sixteen children with S. dysenteriae 1 infection had leukemoid reaction or hemolytic uremic syndrome (complicated shigellosis), while the others did not (uncomplicated shigellosis). Stool IL-6 and TNF-alpha concentrations were higher in children with uncomplicated shigellosis than in children with complicated shigellosis (P = 0.009 and < 0.001, respectively) or in uninfected children (P < 0.001). It is concluded that complicated infection is not associated with higher concentrations of the proinflammatory cytokines IL-6 and TNF-alpha in stool.