Takashi Ohkawa

The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses

Summary: The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells. Hepatocytes produce acute phase proteins and complement in bacterial infections. Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)‐12. IL‐12 and other monokines (IL‐18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon‐g and thereby acquire cytotoxicity against tumors and microbe‐infected cells. These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases. However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place. Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury. NK1.1 Ag+ T cells and another T‐cell subset with an intermediate T‐cell receptor, CD122+CD8+ T cells, can develop independently of thymic epithelial cells. Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein. NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease.

Decrease of CD56+T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma

CD56+T cells and CD56+natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti‐CD3 Ab, IL‐2, or a combination of IL‐2 and IL‐12 and their IFN‐γ production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (−) patients contained 20% CD56+T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN‐γ production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH‐7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (−) patients. CD56+T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH‐7 cells. These results suggest that a decreased number of CD56+T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56− CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)‐type T cells) and compared them with those of normal CD56− CD57− CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti‐CD3 antibodies, both NK‐type T cells produced much larger amounts of interferon‐γ (IFN‐γ) than CD8+ T cells. Both NK‐type T cells also acquired a more potent cytotoxicity against NK‐sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK‐resistant Raji cells. After the stimulation with a combination of interleukin (IL)‐2, IL‐12 and IL‐15, the IFN‐γ amounts produced were NK cells ≥ CD56+ T cells ≥ CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells ≥ CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells ≥ CD8+ T cells ≥ NK cells. However, the CD3‐stimulated proliferation of both NK‐type T cells was smaller than that of CD8+ T cells partly because NK‐type T cells were susceptible to apoptosis. In addition to NK cells, NK‐type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3‐stimulated IFN‐γ production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK‐type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.

The Mechanism of a Defective IFN-γ Response to Bacterial Toxins in an Atopic Dermatitis Model, NC/Nga Mice, and the Therapeutic Effect of IFN-γ, IL-12, or IL-18 on Dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-γ levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-γ response to staphylococcal enterotoxin B was correlated to the lack of regular Vβ8+ T cells and Vβ8+ NK T cells, and the low IFN-γ response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-γ, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

Response to interferon therapy in children with chronic hepatitis C.

We evaluated the effect of interferon therapy and investigated factors that may influence the outcome in 18 children with chronic hepatitis C. A complete response was obtained in 10 children (56%). The titer for pretreatment serum virus ribonucleic acid (< or = 10(7) copies/ml) was correlated significantly with a complete response to therapy.

Mouse CD8+ CD122+ T Cells with Intermediate TCR Increasing with Age Provide a Source of Early IFN-γ Production

Although CD8+ IL-2Rβ (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rβ-negative or -low (CD122−) T cells conversely decreased. The IFN-γ production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-γ than did CD8+ CD122− T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-γ production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-γ production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-γ. The CD3-stimulated IFN-γ production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-γ than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-γ production and are suggested to be involved in the immunological changes with aging.

Functional and Vβ repertoire characterization of human CD8+ T‐cell subsets with natural killer cell markers, CD56+ CD57− T cells, CD56+ CD57+ T cells and CD56− CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK‐type T cell); CD56 single positive (CD56)‐T cells, CD56/CD57 double positive (DP)‐T cells and CD57 single positive (CD57)‐T cells in the peripheral blood. All NK‐type T‐cell populations expressed CD122 and intermediate levels of T‐cell receptor (TCR; regular CD8+ T cells are CD122− and express high levels of TCR). The number of both DP‐T cells and CD57‐T cells, but not CD56‐T cells, gradually increased with age. All NK‐type T‐cell populations produced larger amounts of interferon‐γ than did regular CD8+ T cells after stimulation with interleukin (IL)‐2, IL‐12 and IL‐15. However, CD56‐T cells and CD57‐T cells but not DP‐T cells showed a potent antitumour cytotoxity to NK‐sensitive K562 cells, whereas only CD56‐T cells showed a potent cytotoxity to NK‐resistant Raji cells. Furthermore, although NK‐type T cells produced large amounts of soluble Fas‐ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VβT cells were found in each NK‐type T‐cell population. The non‐variant CDR3 region(s) for the TCRβ chain(s) showed CD57‐T cells and CD56‐T cells to be derived from distinct origins, while the DP‐T cell population consisted of a mixture of the clones seen in both CD56‐T cells and CD57‐T cells. Our results suggest that CD57‐T cells and CD56‐T cells are functionally and ontogenically different populations while DP‐T cells appear to originate from both CD56‐T cells and CD57‐T cells.

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18

We have reported recently that mouse liver NK cells and NK1·1+ T cells were activated by bacterial superantigens via the IL‐12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK‐type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN‐γ and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR– NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN‐γ production from PBMC. When purified NK‐type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN‐γ, while the IFN‐γ amounts produced by both NK‐type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK‐sensitive K562 cells, whereas both NK‐type T cells showed a more potent cytotoxicity against NK‐resistant Raji cells than did NK cells. The IFN‐γ production from each population as well as from whole PBMC was greatly inhibited by anti‐IL‐12 antibody but not by anti‐IL‐18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti‐IL‐12 antibody while the SEA‐induced proliferation of PBMC was not affected by anti‐IL‐12 antibody. Furthermore, SEA‐activated NK‐type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK‐type T cells are thus involved in bacterial superantigen‐induced immune response.

Effect of weight loss on T-cell receptor-mediated T-cell function in elite athletes.

PURPOSE Long-term intensive exercise by athletes may sometimes lead to a susceptibility to infections. In the present study, we examined the differences in immune function between amateur wrestlers experiencing weight loss (WL) and those without WL who underwent similar intensive exercise training. METHODS Eighteen elite amateur wrestlers who attended the Japanese national championship were classified into two groups. One group consisted of those with either slight or no WL (without WL) (<4%; mean, 1%) (N = 9), and the other group consisted of those who needed a significant WL (with WL) (> or = 4%; mean, 7%) (N = 9) during a 1-month period of intensive training. The leukocyte counts as well as the leukocyte subsets in the peripheral blood were examined. The proliferation and cytokine production in T lymphocytes in response to bacterial superantigens (staphylococcal enterotoxin B, streptococcal pyrogenic exotoxin A) and anti-CD3 antibody (Ab) were also examined. RESULTS The total leukocyte counts and leukocyte subsets did not differ substantially between the groups and were also not different from the findings before starting the intensive exercise training. Natural killer cells and T cells among the lymphocytes significantly increased in both groups, whereas the increase in each group was not different. Although the T-cell responses to bacterial superantigens were not different, the anti-CD3 Ab-stimulated proliferation and interferon-gamma production of lymphocytes from the wrestlers with WL were significantly lower than those of the wrestlers without WL. This hyporesponsiveness to CD3 stimulation recovered 2 months after the tournament when the wrestlers reverted to their normal weight. CONCLUSION Intensive exercise in athletes accompanied by a rapid WL was found to compromise the CD3/T-cell receptor-mediated T-cell function in athletes.

Miller Fisher syndrome with transient coma: comparison with Bickerstaff brainstem encephalitis

We herein report a 4-year-old boy with Miller Fisher syndrome (MFS) who presented with transient coma in addition to the typical triad of internal and external ophthalmoplegia, cerebellar ataxia and areflexia after an influenza type B infection. The electroencephalogram findings revealed intermittently generalized slow wave bursts. The cerebrospinal fluid revealed high protein and a lack of any cellular response. The serum anti-GQ1b IgG antibody was elevated in the acute phase and disappeared in the convalescent phase. The transient coma with the triad of MFS in this patient indicated an extended brainstem lesion including a reticular formation, which is also the responsible lesion of Bickerstaff brainstem encephalitis (BBE), but the magnetic resonance imaging repeatedly showed no abnormal finding. Our patient suggested the involvement of central nervous system in addition to the peripheral nerve injury in MFS. He also suggested that MFS and BBE may belong to the same group of disorders as syndrome of ophthalmoplegia, ataxia and areflexia (SOAA).