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Chemokines and their receptors play critical roles in the selective recruitment of various subsets of leukocytes. Recent studies have indicated that some chemokine receptors are differentially expressed on Th1 and Th2 cells. However, available data concerning the presence of T cells with a Th1 or a Th2 character and the expression of chemokine receptors on infiltrating T cells in the rheumatic joint are still limited. In this study, we investigated the expression of CC chemokine receptor 4 (CCR4) and CCR5, which have been shown to be preferentially expressed on T22 and Th1 respectively on T cells from rheumatoid arthritis (RA) patients. Although both CCR5+ and CCR4+ CD4+ T cell populations were observed in peripheral blood mononuclear cells from healthy controls and osteoarthritis patients, these cell populations were decreased in patients with active RA. In contrast, the vast majority of synovial fluid (SF) T cells from active RA patients expressed CCR5 but not CCR4. CCR5 ligands, MIP-1α and RANTES, were found in RA SF at high levels. CCR5+ CD4+ T cells from SF mononuclear cells of RA patients produced IFN-γ but not IL-4 in response to anti-CD3 stimulation in vitro. These results indicated that differential expression of chemokine receptors plays a critical role for selective recruitment of pro-inflammatory T cells into the joints of RA. [ABSTRACT FROM PUBLISHER]

The high-affinity IgE receptor (FcεRI) and the low-affinity IgG receptor (FcγRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common β subunit (FcRβ) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRβ is essential for the cell surface expression of the FcεRI. In humans, the FcRβ gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRβ in vivo still remains ambiguous. To elucidate the functions of FcRβ, we developed the mice lacking FcRβ [FcRβ(-/-)]. The FcRβ(-/-) mice lacked the expression of the FcεRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRβ(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRβ(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRβ accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues. [ABSTRACT FROM PUBLISHER]

The interaction between CD28 and its ligands, CD80 and CD86, is crucial for an optimal activation of antigen-specific T cells. However, the requirement of CD80 or CD86 co-stimulation in Th2 cell differentiation and activation is controversial. Freshly isolated murine CD4+ and CD8+ T cells were incubated with P815 transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb. Both CD80 and CD86 co-stimulated the proliferation of CD4+ and CD8+ T cells at comparable time-kinetics and magnitude, but CD86 alone was able to co-stimulate IL-4 and especially IL-10 production in CD4+ T cells. In typical Th2-dependent immune responses elicited by Nippostrongylus brasillensis infection, the anti-CD86 mAb treatment but not the anti-CD80 mAb treatment efficiently inhibited antigen-specific IgE and IgG1 production, which was accompanied with the reduced IL-4 production. Our results suggest that CD86 co-stimulation plays a dominant role not only in the primary activation of Th2 cells but also in the secondary interaction between antigen-primed Th2 cells and B cells. [ABSTRACT FROM AUTHOR]

Translocator protein (TSPO) is a mitochondrial outer membrane protein expressed on a variety of immune cells, including macrophages, dendritic cells, and T cells, in addition to neurons and steroid-producing cells. Previous studies of TSPO ligands have suggested that TSPO is involved in multiple cellular functions, including steroidogenesis, immunomodulation, and cell proliferation. Currently, there are limited reports on the effects of TSPO or TSPO ligands on T cell-mediated immune responses. We here investigated the involvement of TSPO/TSPO ligand in T cell responses using a 2,4-dinitro-1-fluorobenzene (DNFB)-induced contact hypersensitivity (CH) model. Treatment with Ro5-4864, a TSPO ligand, during DNFB sensitization reduced the number and activation status of CD4+ and CD8+ T cells in draining lymph nodes and alleviated skin inflammation after DNFB challenge. Adoptive transfer of Ro5-4864-treated mouse-derived DNFB-sensitized T cells to naïve mice inhibited CH responses after DNFB challenge. Ro5-4864-treated sensitized T cells showed lower proliferative responses when stimulated with DNFB-pulsed antigen-presenting cells compared to control-treated sensitized T cells. Ro5-4864 also suppressed cell proliferation, as well as adenosine triphosphate and lactate production, during T cell activation. Moreover, the inhibitory effects of Ro5-4864 on T cell responses were conserved in TSPO-deficient cells. Our results suggest that Ro5-4864 inhibits CH responses by suppressing energy metabolism, at least via glycolysis, to reduce the T cell primary response in a TSPO-independent manner., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene., (© The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.

Rejection of solid organ allografts is promoted by T cells. Recipient T cells can directly recognize intact allo-MHC molecules on donor cells and can also indirectly recognize processed donor-derived allo-peptides presented by recipient antigen-presenting cells in the context of self-MHC molecules. Although CD4(+) T cells primed through the indirect allorecognition pathway alone are sufficient to promote acute allograft rejection, it is unknown how they can mediate graft destruction without cognate recognition of donor cells. In this study, we analyzed the indirect effector mechanism of skin allograft rejection using a mouse model in which SCID recipients bearing MHC class II-deficient skin allografts were adoptively transferred with CD4(+) T cells. Histologically, entire graft necrosis was preceded by mononuclear cell infiltration in the graft epithelia with epithelial cell apoptosis, indicating cell-mediated cytotoxicity against donor cells as an effector mechanism. Beside CD4(+) T cells and macrophages, NK cells infiltrated in the rejecting grafts. Depletion of NK cells as well as blocking of the activating NK receptor NKG2D allowed prolonged survival of the grafts. Expression of NKG2D ligands was up-regulated in the rejecting grafts. These results suggest that NK cells activated through NKG2D contribute to the skin allograft rejection promoted by indirectly primed CD4(+) T cells.

Runx family proteins play indispensable roles in the development of various hematopoietic lineage cells. However, their function in NK cells is still uncertain. We found that NK cells and CD8 T cells dominantly express Runx3 protein, whereas NKT cells and CD4 T cells express Runx1. Reverse transcription-PCR analysis revealed that Runx3 expression is initiated at the NK precursor stage and is maintained along the course of NK cell differentiation. In order to examine their role in the earlier stage of NK cell development, we introduced Runx dominant-negative (Runx dn) form into Lin(-)c-kit(+)Sca-1(+) hematopoietic stem cells, which were applied to NK cell-inducing culture. Post-cultured cells showed a decreased expression of IL-2/IL-15 common receptor beta subunit (CD122), consistent with another finding that Runx binds to promoter region of CD122 gene. To examine the Runx function in the later developmental stage, we used transgenic mouse, in which Runx dn form is expressed in immature and mature NK cells. This mouse showed decreased expressions of NK maturation markers, such as Ly49 family, Mac-1 and CD43, whereas IFN-gamma production was greatly enhanced. These findings suggest that Runx proteins, especially Runx3, play multiple roles in NK cell differentiation.

Rheumatoid arthritis (RA) is characterized by hypertrophic synovial tissues comprising excessively proliferating synovial fibroblasts and infiltrating inflammatory cells. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, inflammation and angiogenesis by acting on various cell types. In RA synovial tissues, TGF-beta is expressed at high levels. However, the precise role of TGF-beta in RA remains unclear. We herein demonstrated a causal link between the TGF-beta-induced RA synovial cell proliferation and induction of platelet-derived growth factor (PDGF)-AA. In addition, TGF-beta induced IL-6 and vascular endothelial growth factor (VEGF) production by RA synovial fibroblasts associated with nuclear factor-kappa B activation. These effects of TGF-beta on RA synovial fibroblasts were suppressed by TGF-beta type I receptor kinase inhibitor HTS466284. Furthermore, HTS466284 significantly prevented anti-collagen type II antibody-induced arthritis in mice according to the clinical manifestations, histology, tumor necrosis factor-alpha, PDGF and VEGF expression and 5-bromo-2'-deoxyuridine incorporation. These in vitro and in vivo results suggest that TGF-beta plays a role in the development of synovial hyperplasia consisting of synovial cell proliferation, inflammation and angiogenesis. The blockade of TGF-beta signaling may thus become an additional strategy for the treatment of RA.

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.

Some epidemiological or association studies suggest that transforming growth factor-beta (TGF-beta) in breast milk may be a decisive factor in diminishing the risk of allergic diseases during infancy. The observations have prompted us to investigate whether TGF-beta, when taken orally, can affect allergic immune responses. Repeated high-dose ovalbumin peptide (OVA) feeding was previously reported to induce OVA-specific IgE production and an anaphylactic reaction after intravenous challenge of OVA in OVA-TCR transgenic mice, which might represent a model for food allergy. By using this model, we showed here that oral administration of high-dose TGF-beta simultaneously with OVA feeding significantly inhibited the OVA-specific IgE elevation and anaphylactic reaction in OVA-TCR transgenic DO11.10 mice. These effects were associated with suppression of OVA-specific IL-4 production and GATA-3 expression and with up-regulation of IFN-gamma production and T-bet expression by splenocytes. Intra-peritoneal injection of anti-TGF-beta-neutralizing antibody abolished the inhibitory effects of orally administered TGF-beta on the serum IgE response and anaphylactic reaction after OVA feeding in DO11.10 mice. Interestingly, oral administration of high-dose TGF-beta suppressed activation-induced T cell death induced by OVA feeding in DO11.10 mice. We thus conclude that TGF-beta, when taken orally at high dose, has the capacity to modulate a food allergy-related reaction, at least in part, through its systemic activity.

Intraocular inoculation of antigens induces anterior chamber-associated immune deviation (ACAID), which is mediated by development of regulatory T cells in response to antigen-presenting cells (APC) pre-conditioned by intraocular transforming growth factor-beta (TGF-beta). In this study, we examined the involvement of T-cell co-stimulatory molecules in this process. To mimic the intraocular APC, thioglycollate-elicited peritoneal exudate cells (PEC) were pre-treated with TGF-beta in vitro. Expression of CD80, CD86, OX40 ligand (OX40L) and CD70 was analyzed by flow cytometry. Contribution of these molecules to co-stimulatory activity of TGF-beta-treated PEC on antigen-stimulated T-cell proliferation and cytokine production was determined by inhibition with blocking antibodies in vitro. Contribution of CD80 and CD86 to induction of ACAID was determined by the administration of blocking antibodies at intraocular antigen inoculation in vivo. TGF-beta-treated PEC expressed CD80 and CD86 but not OX40L or CD70. Antigen-stimulated T cells proliferated and produced IL-10, but not IFN-gamma, in response to co-stimulation by TGF-beta-treated PEC, which was abrogated by blocking antibodies against CD80 and CD86. Induction of regulatory cells mediating ACAID was abolished by in vivo blockade of CD80 and CD86. The present results indicated that CD80 and CD86 play a critical role in induction of ACAID, possibly by co-stimulating expansion and IL-10 production of regulatory T cells in response to TGF-beta-conditioned APC.

Inoculation with erythrocytes infected with Plasmodium berghei, a protozoan causing mouse lethal malaria, induces liver injury in mice, although the parasite cannot invade host hepatocytes at this infectious stage. As previously reported, hepatic infiltrates participate in this liver injury by exerting their perforin-dependent killing action. Here, we have investigated the cellular mechanisms underlying P. berghei-induced incidental liver injury. Hepatic lymphocytes from P. berghei-infected mice killed normal hepatocytes, but not ConA-induced lymphoblasts. Furthermore, the hepatic lymphocytes from infected C57BL/6 mice displayed cytotoxicity against hepatocytes from C57BL/6 and BALB/c mice, indicating MHC-unrestricted hepatocytotoxicity by these hepatic lymphocytes. NK cells were not involved in this hepatocytotoxicity. However, DX5+ cells sorted from the liver of infected CD1d-deficient mice killed normal hepatocytes, indicating that CD1d-independent DX5+ T cells are the effector cells. The hepatocytotoxicity of these hepatic DX5+ T cells did not require TCR engagement. These findings indicate that hepatic CD1d-independent DX5+ T cells play a critical role in P. berghei-induced liver injury. Our studies may have general implications for tissue injuries that are caused by 'bystander killing' or other poorly defined cell-mediated killing mechanisms., (Copyright 2004 The Japanese Society for Immunology)

The beta chain, a component of the high-affinity receptor for IgE (FcepsilonRI), plays an important role in IgE-mediated allergic reaction. The beta chain accelerates the function of FcepsilonRI by amplification of its surface expression and of signal transduction in effector cells such as mast cells and basophils. Two regulatory regions, +60/+66 and +70/+76, for the human beta chain gene that are required for the cell-type-specific transcriptional activation were identified by transient reporter assay. Electrophoretic mobility shift assay demonstrated that Oct-1 binds both the regions, among which the +70/+76 Oct-1 motif was critical for the transactivation of the beta promoter responsive to Oct-1 overexpression. Regulation of beta chain gene expression is discussed.

Induction of alphaEbeta7 expression on T cells by transforming growth factor (TGF)-beta is thought to be important for intestinal intraepithelial T lymphocyte (IEL) entry into the epithelial compartment. However, there has been no in vivo evidence that up-regulation of alphaEbeta7 expression on T cells by TGF-beta is critical for the selective localization of intestinal IEL in the epithelial area. We have recently established transgenic mice expressing Smad7 under the control of a distal lck promoter where TGF-beta/Smad signaling is specifically blocked in mature T cells. Here we showed that TGF-beta-mediated up-regulation of alphaEbeta7 was impaired on T cells isolated from the Smad7 transgenic mice associated with reduced numbers of intestinal IEL when compared with that in wild-type littermates. These results indicated that failure to induce alphaEbeta7 on T cells by TGF-beta resulted in reduced numbers of intestinal IEL, suggesting the importance of alphaEbeta7 expression by TGF-beta in selective localization of intestinal IEL.

4-1BB (CDw137) and its ligand (4-1BBL) have been implicated in cellular immune responses. To further characterize the expression and function of 4-1BBL, we newly generated an anti-mouse 4-1BBL mAb (TKS-1), which can inhibit the interaction of 4-1BBL with 4-1BB. Flow cytometric analyses using TKS-1 and an anti-mouse 4-1BB mAb indicated that 4-1BB was inducible on both CD4(+) and CD8(+) splenic T cells by stimulation with immobilized anti-CD3 mAb, but 4-1BBL was not expressed on resting or activated T cells. 4-1BBL expression was inducible on splenic B cells by stimulation with anti-IgM antibody plus anti-CD40 mAb, on peritoneal macrophages by stimulation with lipopolysaccharide (LPS) and on splenic dendritic cells (DC) by stimulation with anti-CD40 mAb or LPS. Interestingly, splenic DC expressed 4-1BB constitutively, which was down-regulated by anti-CD40 stimulation. Co-culture of splenic DC with 4-1BBL-transfected cells or 4-1BBL-expressing tumor cell lines led to cytokine (IL-6 and IL-12) production and co-stimulatory molecule up-regulation by splenic DC, indicating that 4-1BBL can directly activate DC. Moreover, IL-12 production by anti-CD40-stimulated DC was partially inhibited by TKS-1. These results suggest that 4-1BB expressed on DC may be involved in DC activation through DC--tumor interaction and DC--DC interaction.

In vascularized organ transplantation, vascular endothelial cells (EC) confronting recipient T cells are potentially significant APC initiating cellular immune responses that lead to rejection. In the present study, we studied the ability of human EC to stimulate allogeneic T cells and the co-stimulatory molecules involved in this response. On both human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MVEC), MHC class I, intercellular adhesion molecule (ICAM)-1 and CD86 were constitutively expressed as assessed by flow cytometry. After IFN-gamma treatment, MHC class II expression was induced, and MHC class I and ICAM-1 were up-regulated. In contrast, the expression of CD86 was unchanged and CD80 was undetectable even after IFN-gamma treatment. Highly purified CD4+ T cells proliferated in response to IFN-gamma-treated allogeneic HUVEC and MVEC, and this response was efficiently blocked by mAb to MHC class II, ICAM-1 and CD86. Furthermore, the addition of anti-CD86 mAb to the primary culture with allogeneic EC resulted in the induction of alloantigen-specific anergy. These results suggest that CD86 expressed on EC plays a critical role in initiating cellular immune responses to vascularized allografts and would be an important target for immune intervention.

We have investigated the involvement of adhesion molecules in the lymphocyte infiltration associated with acute intestinal graft-versus-host disease (GVHD) induced by injection of C3H lymph node cells into irradiated (C3H x DBA/2)F1 mice. First we analyzed the expression profile of adhesion molecules including alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, alpha L and beta 7 integrins, CD44 and L-selectin of lymphocytes from lymph nodes and gut mucosa in normal mice. In normal mice, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) uniquely showed increased expression of alpha 1, alpha 2 and beta 7 integrins, and decreased expression of L-selectin compared with that of lymphocytes of the lymph nodes and Peyer's patches. In mice with GVHD, IEL and LPL of donor lymph node cells origin underwent phenotypic changes characterized by the increased expression of alpha 1, alpha L and beta 7 integrins, and the loss of L-selectin. The expression profile of adhesion molecules on IEL and LPL of GVHD mice resembled that of normal mice except for the lack of alpha 2 integrin. Treatment of GVHD mice with anti-alpha 1, -alpha 4 or -beta 7 integrin antibody alone partially prevented the mucosal pathology of intestinal GVHD, whereas only mice treated with anti-alpha 1 showed reduced donor lymphocytic infiltration into the intestinal mucosa. In contrast, treatment with anti-alpha L or anti-CD44 antibody did not affect the intestinal GVHD. Furthermore, dual blockade of both alpha 1 and alpha 4 integrins completely inhibited the mucosal pathology and donor lymphocyte infiltration of intestinal GVHD.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta 1 integrin subfamily, alternatively called very late activation antigen (VLA), has been implicated in various cellular functions. In this study, we generated a mAb against the mouse beta 1 subunit (CD29) to examine the functional property of mouse VLA proteins. After immunization with affinity-purified mouse VLA-4 (alpha 4 beta 1), a hamster mAb, HM beta 1-1, was established by screening mAb that reacted with alpha 4-negative neuroblastoma C1300. The antigen defined by HM beta 1-1 was widely distributed in various mouse cell lines and HM beta 1-1 immunoprecipitated a 110-120 kDa protein common to VLA-1 and VLA-6, indicating that HM beta 1-1 recognizes the beta 1 subunit of mouse integrins. We then examined the inhibitory effect of HM beta 1-1 on VLA-dependent cell adhesion and activation. HM beta 1-1 blocked the adhesion of mouse tumor cell lines to extracellular matrix proteins including collagen, laminin and fibronectin. Moreover, splenic T cell proliferation induced by anti-CD3 mAb and allogeneic mixed lymphocyte response were strongly inhibited by HM beta 1-1 in combination with an anti-LFA-1 mAb. We conclude that HM beta 1-1 reactive with mouse CD29 can inhibit VLA-dependent cellular functions and, thus, would be useful for studying the physiological role of beta 1 integrins in vivo.

Adhesion molecules of the integrin family are implicated not only in leukocyte migration but also in leukocyte activation. Here we characterize the expression and function of fibronectin receptor integrins on rat mast cells. A rat basophilic leukemia cell line (RBL-2H3) and phorbol ester-stimulated rat peritoneal mast cells adhered to fibronectin (FN), vitronectin and fibrinogen. These mast cells expressed fibronectin receptor integrins, including very late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR), as estimated by immunofluorescent staining and inhibition of FN adherence by newly established mAbs reactive with the rat alpha 4 (MR alpha 4-1), alpha 5 (HM alpha 5-1) or beta 3 (HM beta 3-1) chains of the integrin molecules. The beta-hexosaminidase release, a marker for mast cell degranulation, triggered by high affinity IgE receptor (Fc epsilon RI)-mediated stimulation, was enhanced by adhesion of RBL-2H3 cells to either immobilized FN, MR alpha 4-1, HM alpha 5-1 or HM beta 3-1. This FN enhancement of beta-hexosaminidase release was inhibited by soluble MR alpha 4-1, HM alpha 5-1 and HM beta 3-1 as well as by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogate VLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passive cutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA was inhibited by concurrent s.c. injection of MR alpha 4-1, HM alpha 5-1 and HM beta 3-1. These results demonstrate that FN receptor integrins expressed on rat mast cells play an important role in regulating mast cell activation both in vitro and in vivo.

Diverse adhesion molecules participate in many important responses and thus would be implicated in the pathogenesis of various autoimmune diseases. However, there is little evidence for the role of these molecules in autoimmune insulin-dependent diabetes mellitus. Here we present several lines of evidence suggesting that leukocyte function-associated antigen-1 (LFA-1) and its counter-receptor intercellular adhesion molecules (ICAM-1), one of the most important pairs among these adhesion molecules, are involved in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse. Immunohistochemical study showed the hyperexpression of ICAM-1 on islet-infiltrating mononuclear cells and vascular endothelium in NOD pancreas. In vivo administration of anti-LFA-1 or anti-ICAM-1 mAb from 5 to 30 (or 12) weeks of age exerted a very strong preventative effect on the development of spontaneous diabetes with a marked reduction of insulitis, whereas both antibodies, even combined to use simultaneously, could not prevent cyclophosphamide-induced diabetes. Adoptive transfer of insulitis and diabetes to young NOD mice following the injection of islet-derived mononuclear cells from diabetic donors was completely blocked by administration of both antibodies to recipients. The present study, therefore, provides the first evidence that immunointervention to LFA-1-ICAM-1 interaction has a strong prophylactic effect on autoimmune diabetes in NOD mice.

The present study aimed to develop an assay system capable of directly examining the adhesion of cells to each cell-binding site on fibronectin (FN) and to investigate molecular mechanisms underlying the pre-B cell-FN interaction. Treatment of culture plates with 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester and subsequently with dithiothreitol (DTT) allowed the plates to adsorb DTT-treated extracellular matrix (ECM) proteins as well as synthetic peptides containing cysteine at the N-terminal. These treatments produced culture plates coated with ECM proteins or peptides corresponding to its cell-binding sequences, i.e. three sites on FN termed RGDS, LDVP, and RGDV. Pre-B cells exhibited potent adhesiveness to FN-coated plates. Its FN binding was most efficiently inhibited by adding a combination of free forms of RGDS, LDVP, and RGDV peptides, indicating the involvement of these three cell-binding sites in the pre-B cell-FN interaction. In accordance with this, pre-B cells exhibited considerable and potent binding to the respective RGDS-, LDVP-, or RGDV-coated plates. Such binding was specific for the peptide used for coating, because each binding to a given peptide-coated plate was inhibited only by addition of a homologous free peptide. This assay system further demonstrated that the pre-B cell binding to RGDS was mediated by the alpha 5 and alpha v integrins, whereas the binding to LDVP and RGDV was mediated by the alpha 4 integrin. It was also shown that LDVP binding was inhibited by LDVP but not by RGDV and, likewise, RGDV binding was inhibited by RGDV but not by LDVP.2+ interaction involving complex molecular mechanisms.

The high-affinity receptor for IgE (Fc epsilon RI) has a tetrameric structure structure composed of one alpha, one beta, and two disulfide-linked gamma subunits, of which the alpha subunit binds IgE with high affinity. A recombinant soluble form of the ectodomain of the human Fc epsilon RI alpha subunit (rsFc epsilon RI alpha) was recently generated by gene engineering and was verified to bind IgE with an affinity as high as that of native Fc epsilon RI on the cell surface. rsFc epsilon RI alpha was prepared on a large scale in order to analyze its biological function. rsFc epsilon RI alpha completely inhibited IgE binding to the cell surface, resulting in abrogation of the chemical mediator release from RBL-2H3 cells. Furthermore it completely abolished the passive cutaneous anaphylaxis (PCA) response by trapping IgE specifically when it was administered into rats prior to IgE sensitization. Even after IgE sensitization, treatment of rsFc epsilon RI alpha substantially reduced the PCA response. It was finally shown that rsFc epsilon RI alpha inhibited IgE binding to human peripheral blood basophils and the histamine release from them. In this paper we address the ability of rsFc epsilon RI alpha to specifically prevent the IgE-mediated allergic reaction.

To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

The killer cell characteristics of Thy-1-positive dendritic epidermal cells (Thy-1+ DEC) were examined. Four Thy-1+ DEC clones which were established from athymic nude mice exhibited spontaneous or lectin-redirectable cytotoxic activity against some murine tumor cell lines in a 4 h 51Cr-release assay. A colorimetric assay for benzyloxycarbonyl-L-lysine-thiobenzyl ester esterase revealed a strong serine esterase activity expressed in all cell clones. In addition, Northern blot analysis using a murine perforin cDNA probe revealed that all four Thy-1+ DEC clones expressed abundant mRNA for perforin, as do most killer T cells. More importantly, immunocytochemical staining with an anti-perforin monoclonal antibody revealed that not only all four Thy-1+ DEC clones but also a part of freshly isolated Thy-1+ DEC from normal and nude mice contained perforin. These results demonstrate that Thy-1+ DEC exhibit typical killer cell characteristics in vitro and in vivo. These data also suggest that Thy-1+ DEC may play a cytotoxic role in protecting the integrity of skin from infection or neoplastic transformation.

CD2 expression in murine B cell lineage was examined by flow cytometric and immunoprecipitation studies with anti-murine CD2 mAb and by Northern blot analysis. Cell surface expression of CD2 was demonstrated on all peripheral B cells and cell lines of B lineage. The murine CD2 transcript of 1.3 kb was detected in these B cells. An identical glycoprotein of 55-67 KD was precipitated from the lysates of surface radioiodinated thymocytes, splenic T and B cells, T and B lymphomas, RL male 1 and BCL-1, with anti-murine CD2 mAb. The majority of bone marrow B cells and a half of pre-B cells were found to be positive for CD2 expression. These results indicate that murine CD2 is expressed on B cell lineage at certain differentiation stages.