Kisaburo Nagata

The Igα/Igβ Heterodimer on μ-Negative ProB Cells Is Competent for Transducing Signals to Induce Early B Cell Differentiation

Abstract The immunoglobulin α (Igα)/Igβ heterodimer was detected on the surface of μ-negative proB cell lines in association with calnexin. The cross-linking of Igβ on proB cells freshly isolated from bone marrow of recombination activating gene (RAG)-2–deficient mice induced a rapid and transient tyrosine-phosphorylation of Igα as well as an array of intracellular proteins including Syk, PI3-kinase, Vav, and SLP-76. It also elicited the phosphorylation and activation of a MAP kinase ERK but not JNK/SAPK or p38. When RAG-2–deficient mice were treated with anti-Igβ monoclonal antibody, developmentally arrested proB cells were induced to differentiate to the small preB cell stage as observed when the μ transgene was expressed in RAG-2–deficient mice. Thus, the cross-linking of Igβ on proB cells appears to elicit differentiation signals analogous to those delivered by the preB cell receptor in normal B cell development.

Induction of superoxide anion production from monocytes and neutrophils by activated platelets through the P-selectin-sialyl Lewis X interaction

Activated platelets expressing P‐selectin on their surface are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes were activated by their adhesion to activated platelets and demonstrated that P‐selectin–dependent platelet adhesion to neutrophils and monocytes induced production of extracellular superoxide anion (O2 ‐) by these leukocytes. Leukocyte membrane glycoproteins containing Ser/Thr‐linked carbohydrate chains were responsible for the signal reception leading to the leukocyte activation. Cytokines were shown to influence these processes. For example, treatments of neutrophils with interleukin‐8 (IL‐8) or granulocyte colony‐stimulating factor (G‐CSF) potentiated the P‐selectin‐induced O2 ‐ production. Furthermore, interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) induced surface expression of P‐selectin on platelets in the presence of a low concentration of thrombin and consequently enhanced their adhesion capacity to leukocytes. These results indicated that the adhesion of activated platelets to the leukocytes through the interaction between P‐selectin and its carbohydrate ligand, sialyl Lewis X (Lex), was a crucial step for the activation of leukocyte function and supported the notion that activated platelets were actively involved in the inflammatory processes. J. Lcukoc. Biol. 56: 583–587; 1994.

Density‐dependent induction of TNF‐α release from human monocytes by immobilized P‐selectin

P‐selectin purified from human platelets, when immobilized on a solid surface, induced monocytes to release tumor necrosis factor‐α (TNF‐α). The induction of TNF‐α release was dependent on the concentration of P‐selectin used for the immobilization, and the maximal stimulation was observed when the plate was coated with 0.3 μg/ml of P‐selectin. Use of either a higher or a lower concentration of P‐selectin for the plate‐coating was found to elicit less TNF‐α release, although the higher concentration of P‐selectin caused a stronger adhesion of HL‐60 leukemic cells. The expression of mRNA for TNF‐α roughly paralleled the TNF‐α secretion, as assessed by RT‐PCR. These results indicate that monocytes are activated by immobilized P‐selectin in a density‐dependent manner.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Neutrophils Accelerate Macrophage-Mediated Digestion of Apoptotic Cells In Vivo as Well as In Vitro

It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In contrast, we previously found that injection of apoptotic cells into the peritoneal cavity induced the expression of an inflammatory chemokine, MIP-2, and infiltration of neutrophils, and that anti-MIP-2 Abs suppressed the infiltration significantly. Because our previous study showed that whole-body x-irradiation caused neutrophil infiltration into the thymus along with T cell apoptosis, we examined the role of neutrophils in apoptotic cell clearance. Neutrophil infiltration reached a peak 12 h after irradiation with 1 Gy of x-rays. Immunohistological analysis revealed that apoptotic cells disappeared dramatically from 10.5 to 12 h after x-irradiation. As neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Either anti-MIP-2 or anti-CXCR2 Abs suppressed neutrophil infiltration significantly, and the suppression of neutrophil infiltration by anti-MIP-2 Abs delayed the disappearance of apoptotic cells. Moreover, macrophage-mediated digestion of apoptotic thymocytes was accelerated in vitro on coculturing with neutrophils, even if neutrophils were separated from macrophages. These results suggest that neutrophils are recruited to the thymus mainly by MIP-2 after whole-body x-irradiation and that such neutrophils may not induce inflammation but rather accelerate complete digestion of apoptotic cells by macrophages.

Cutting edge : A critical role of nitrogen oxide in preventing inflammation upon apoptotic cell clearance

Apoptotic cells are removed by phagocytes without causing inflammation. It remains largely unresolved whether anti-inflammatory mediators prevent neutrophil infiltration upon apoptotic cell clearance in vivo. In this study, we showed that, upon induction of apoptosis in the thymus by x-ray, inducible NO synthase knockout (KO) mice exhibited higher levels of neutrophil infiltration and production of MIP-2 and keratinocyte-derived chemokine (KC) in the thymus than wild-type (WT) mice. Furthermore, administration of NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase, to x-irradiated WT mice increased the level of neutrophil infiltration to that of KO mice by the augmentation of MIP-2 and KC production. Additionally, thymic macrophages isolated from x-irradiated KO mice produced more MIP-2 and KC than those from WT mice. Thus, although apoptosis is believed to be noninflammatory, this is actually achieved by the production of immunosuppressive signals such as NO that counteract proinflammatory chemokines such as MIP-2 and KC.

Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a mucin‐like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet‐leukocyte and endothelium‐leukocyte interactions. The treatment of neutrophils with a low concentration of IL‐8 induced the redistribution of PSGL‐1 to one end of the cell to form a cap‐like structure. We investigated the role of lipid microdomains in the redistribution of PSGL‐1 and its effect on the adhesive characteristics of IL‐8‐treated neutrophils. The redistribution of PSGL‐1 induced by IL‐8 was inhibited by cholesterol‐perturbing agents such as methyl‐β‐cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL‐1 was enriched in a low‐density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X‐100 was used for the solubilization, PSGL‐1 was no longer recovered in the low‐density fraction, although GM1 ganglioside remained in the low‐density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL‐1 differed from that of GM1 ganglioside, suggesting that PSGL‐1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL‐8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross‐linking of PSGL‐1 with antibodies. These results suggest that the association of PSGL‐1 with lipid microdomains is essential for its redistribution induced by IL‐8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P‐selectin.

Regulation of the estrous cycle by neutrophil infiltration into the vagina

During metestrus of the estrous cycle, a number of neutrophils infiltrate into the vaginal vault, presumably due to a neutrophil-specific chemokine, MIP-2, in mice. The physiological role of the infiltrating neutrophils, however, remains largely obscure. In this study we examined the effects of neutrophil depletion on the estrous cycle and steroid hormone levels. When mice were treated with an anti-Gr-1 mAb, they became neutropenic, as assessed as to the number of neutrophils in the peripheral blood. The estrous cycle of such mice was specifically blocked at diestrus irrespective of the phase at which the anti-Gr-1 mAb was administered. The blockade was reversible, because restoration of neutrophils to a normal level caused a restart of the cycle. Immunohistochemical analyses revealed that neutrophils were present mainly on the luminal surface and in the lumen at metestrus and to a lesser extent at diestrus but scarcely in the uterine cervix at any phase, and that the anti-Gr-1 mAb depleted neutrophils but not eosinophils in the vagina. The treatment with the anti-Gr-1 mAb significantly affected the serum 17beta-estradiol and progesterone levels at diestrus after the estrous cycle was blocked. Together, these results suggest that neutrophil infiltration into the vagina is critical in maintaining the estrous cycle through control of steroid hormone levels.

Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes

Lymphocyte‐oriented kinase (LOK) is a member of the STE20/p21‐activated kinase (PAK) family and expressed predominantly in lymphoid organs. Generation of LOK‐deficient mice revealed that the leukocyte‐function‐associated antigen (LFA‐1)/intercellular adhesion molecules (ICAM)‐mediated aggregation of mitogen‐stimulated T cells was greatly enhanced in the absence of LOK. Though levels of total LFA‐1 and ICAMs as well as the active form of LFA‐1 on T cell blasts were comparable in the presence and absence of LOK, clustering of active LFA‐1 detected by binding of soluble ICAM‐1 was accelerated in the absence of LOK. These results suggest that LOK is potentially involved in the regulation of LFA‐1‐mediated lymphocyte adhesion.

Infiltrating neutrophils induce allospecific CTL in response to immunization with apoptotic cells via MCP-1 production

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity. As neutrophils are known to function as effectors as well as regulators in the immune response, we examined the roles of infiltrating neutrophils in alloantigen‐specific CTL induction after immunization with late apoptotic P388 cells. The CTL induction and infiltration of CD8+ T cells into the peritoneal cavity were inhibited by depletion of neutrophils by anti‐Gr‐1 mAb or inhibition of neutrophil infiltration by anti‐MIP‐2 antibody, suggesting that neutrophils are involved in CD8+ T cell infiltration into the peritoneal cavity. It is known that MIP‐1α, MIP‐1β, and MCP‐1 are capable of attracting CD8+ T cells and that they are produced by neutrophils. These chemokines were detected in the peritoneal cavity, and among them, MCP‐1 production was reduced remarkably by suppression of neutrophil infiltration. Moreover, infiltration of CD8+ T cells into the peritoneal cavity as well as CTL activity was clearly reduced by administering anti‐MCP‐1 antibody i.p. Furthermore, the CTL induction and infiltration of CD8+ T cells in neutrophil‐depleted mice were restored significantly by administering recombinant murine MCP‐1 into the peritoneal cavity. These results indicate that MCP‐1 appears to link infiltration of neutrophils with CTL induction.