Hoshio Hiraide

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.

Nuclear p53 immunoreaction associated with poor prognosis of breast cancer

p53 protein has been frequently detected at high levels in the nuclei of human breast cancer cells. We analyzed inununohistochemically the association between nuclear localization of p53 protein and clinical and histological parameters of breast cancer patients. Surgically resected tissues of 73 primary breast cancers were processed by acetone fixation and paraffin embedding and examined using an anti‐p53 monoclonal antibody, Pabl801. p53 immunoreactivity was detected in the nuclei of cancer cells in 17 cases (23%). The nuclear p53 immunoreaction was closely associated with overexpression of c‐erbB‐2 protein (P<0.05), high histologic grade (P<0.01), advanced clinical stage (P<0.05), and negative estrogen receptor status (P< 0.01). When 31 cases which had been followed up for more than 50 months were examined, a positive nuclear p53 immunoreaction was found to he significantly associated with shorter overall survival of patients (P<0.01). These results suggest that inununohistochemical examination of nuclear p53 protein is clinically useful as an indicator of breast cancer aggressiveness.

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.

Neutrophil elastase, MIP-2, and TLR-4 expression during human and experimental sepsis

Highly activated neutrophils play a critical role in mediating organ injury in sepsis by releasing neutrophil elastase (NE). Toll-like receptors (TLRs) play an important role in the host defense against invading microbes, and their signaling pathway is critical to the activation of the proinflammatory response. However, the relationship between TLR expression and the host defense mechanism during sepsis has not been fully elucidated. In this paper, we investigated the relationships among chemokine (MIP-2), TLR-4, and NE expression in human sepsis and murine peritonitis (CLP). TLR-4 expression on monocytes/macrophages was examined in patients with sepsis and in murine peritonitis and was markedly increased in both populations. LPS-induced MIP-2 production by bronchoalveolar cells and liver mononuclear cells in mice with peritonitis was also significantly increased compared with sham-operated mice. Pretreatment of the macrophage cell line, RAW 264.7 cells, with a NE inhibitor before their exposure to LPS resulted in a significant dose-dependent decrease in MIP-2 production, which was comparable to that seen following pretreatment with TLR-4 antibody. Furthermore, NE and LPS both up-regulated TLR-4 expression on human peripheral blood monocytes. Thus, chemokine-induced recruitment of neutrophils in sepsis may result in further increased chemokine production and increased expression of TLR-4. Neutrophil-derived NE may be associated with increased expression of monocyte/macrophage TLR-4, thereby serving as a positive feedback loop for the inflammatory response among the different cell populations.

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.

Activation of Monocytes and Endothelial Cells Depends on the Severity of Surgical Stress

Surgical injury not only induces a systemic endocrine-metabolic response but also influences the function of the leukocytes and endothelial cells leading to various systemic responses. These responses appear to depend on the severity of surgical stress, which differs according to the surgical procedures. In this study, we investigated the response of monocytes and endothelial cells, and the development of systemic inflammatory response syndrome (SIRS) in relation to the severity of surgical stress. The postoperative clinical course was evaluated between patients undergoing an esophagectomy (ER group) and a distal gastrectomy (DG group). The tumor necrosis factor α (TNFα) production of monocytes, the serum interleukin 6 (IL-6) levels, the CD11b expression on either monocytes or granulocytes, and the intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (HUVECs) stimulated with culture supernatants of monocytes were compared between the 2 groups. The development of SIRS was observed in all patients in the ER group, whereas no patients demonstrated SIRS in the DG group. The serum IL-6 levels, TNFα production of monocytes, and CD11b intensity on monocytes or granulocytes in the ER group were higher than those in the DG group. In the ER group, the ICAM-1 intensity on HUVECs with monocytes immediately after operation significantly increased compared with before the operation. In conclusion, both the CD11b expression on monocytes and the TNFα production of monocytes are considered to reflect the degree of surgical stress, and the activation of endothelial cells stimulated with these activated leukocytes may therefore lead to both tissue and organ injury.

Correlation of KIT and EGFR overexpression with invasive ductal breast carcinoma of the solid‐tubular subtype, nuclear grade 3, and mesenchymal or myoepithelial differentiation

Although KIT and EGFR overexpressions are reported to occur in breast cancer, their pathological significance is still unclear. We examined KIT, EGFR, and c‐erbB‐2 overexpressions immunohistochemically in 150 cases of surgically resected breast cancer and their correlation with the histological type and grade and mesenchymal and/or myoepithelial immunophenotype of primary tumors. To facilitate the analysis, we constructed a tissue microarray comprising 2‐mm diameter tissues cored from the representative tissue block of each tumor. KIT, EGFR, and c‐erbB‐2 overexpressions were detected in 15 (10%), 12 (8%), and 23 (15%), respectively. The KIT was more frequent in the group comprising comedo‐type ductal carcinoma in situ and invasive ductal carcinomas (IDCs) of the solid‐tubular subtype than in the group of other histological types (P = 0.027), and the EGFR was more frequent in IDCs of solid‐tubular type than in other histological types (P < 0.05). KIT and EGFR overexpressions were correlated with nuclear grade 3 (P = 0.0095 and 0.0005) and tended to be concurrent (P = 0.005). KIT overexpression was correlated with vimentin and S‐100 expression (P = 0.003 and P = 0.005), and EGFR overexpression was correlated with S100 expression (P = 0.0001). These correlations with grade and mesenchymal/myoepithelial markers were not observed for c‐erbB‐2 overexpression. KIT and EGFR appeared to be indicators of high‐grade breast carcinoma groups that often contain the carcinomas with mesenchymal and/or myoepithelial differentiation, which are distinct from the group with c‐erbB‐2 overexpression. (Cancer Sci 2005; 96: 48 –53)

Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice

Depletion of both natural killer 1.1+ (NK1+) intermediate αβ T‐cell receptor (int T) cells and NK cells by in vivo treatment with anti‐NK1 antibody greatly increased hepatic metastases of intravenously injected EL4 cells as well as pulmonary metastases of 3LL cells in C57BL/6 mice. However, depletion of NK cells alone by anti‐asialo GM1 (AGM1) antibody treatment did not increase the metastases in either organ. Interleukin‐12 (IL‐12) administration into mice induced strong cytotoxicities of NK cell‐depleted liver and lung mononuclear cells (MNC) comparable to those without NK‐cell depletion and inhibited metastases in either organ. In contrast, in both NK cell‐ and NK1+ int T‐cell‐depleted mice, IL‐12 could not induce cytotoxic activity of liver and lung MNC and metastases in both organs increased with or without IL‐12 treatment. These results confirmed the fact that NK1+ int T cells are more potent antitumour effectors than NK cells against experimental haematogenous tumour metastases.

Primary small cell (oat cell) carcinoma of the breast: Report of a case and review of the literature

A case of primary small cell (oat cell) carcinoma of the breast in a 41‐year‐old woman is presented. The patient was alive and well without disease 16 months after modified radical mastectomy and subsequent chemotherapy. The tumor cells revealed morphologic similarity to oat cell carcinoma of the lung and immunohistochemical expression of neuroendocrine markers. In ultrastructural examination, the tumor cells had neurosecretory granules. Review of nine previously reported cases and this case of primary small cell carcinoma of the breast has revealed that this type of tumor shows prominent vascular invasion, frequent lymph node metastasis, infrequent expression of estrogen receptor, and also very poor prognosis. Immunohistochemical study for the c‐kit proto‐oncogene product, which has been reported to be a specific marker for pulmonary small cell carcinoma, demonstrated positive reactivity in approximately 80% of the tumor cells of this case, which is the first report according to our knowledge. The expression of c‐kit might be some aid to the diagnosis of primary small cell carcinoma of the breast.