Chikako Iwabuchi

Tumour necrosis factor-α but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation

Using murine spleen‐derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T‐cell (Th) differentiation were analysed. Immature DC (iDC) with high‐ or low‐dose OVA323–339 predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor‐α (TNF/DC) induced a significantly higher Th2 response in the presence of low‐dose OVA323–339 than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high‐dose OVA323–339, LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low‐dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high‐dose antigen to Th1 polarization were not related to the amount of IL‐12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low‐dose antigen are potent inducers of Th2 differentiation.

Amelioration of Experimental Autoimmune Uveoretinitis by Pretreatment with a Pathogenic Peptide in Liposome and Anti-CD40 Ligand Monoclonal Antibody

We have defined a peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to residues 201–216 of bovine interphotoreceptor retinoid-binding protein and induces experimental autoimmune uveoretinitis (EAU)4 in H-2Ak-carrying mice (H-2Ak mice). In this study, we attempted to ameliorate EAU in the H-2Ak mice without nonspecific suppression of T cell responses. Preceding s.c. administration of liposomes including K2 (liposomal K2) specifically inhibited subsequent generation of T cell response to K2. The same result was obtained with a combination of OVA323–339 peptide and the OVA-specific TCR-transgenic T cells. It was suggested that the inhibition was mainly attributed to peripheral anergy induction of T cells specific for the peptide Ag, although specific cell death might also be involved in the inhibition. Pretreatment with liposomal K2 also considerably abolished IFN-γ production but not IL-4 production. The specific inhibitory effect of the pretreatment with liposomal peptide was augmented by a simultaneous administration of anti-CD40 ligand (anti-CD40L) mAb. Moreover, it was shown that the pretreatment with liposomal K2 reduced both the incidence and severity of the subsequent K2-induced EAU, and the simultaneous administration of anti-CD40L mAb augmented this preventive effect by liposomal K2. Our findings demonstrate that the s.c. administration of liposomal pathogenic peptide and anti-CD40L mAb can be applied to preventing autoimmune diseases without detrimental nonspecific suppression of T cell responses.

Immunoregulatory defects of Vα24+Vβ11+ NKT cells in development of Wegener's granulomatosis and relapsing polychondritis

The frequency of either CD4–8– (double negative; DN) or CD4+ Vα24+Vβ11+ NKT cells, the expression of CD1d and the binding of CD1d‐tetramer loaded with α‐galactosylceramide (α‐GalCer) to NKT cells were analysed in peripheral blood mononuclear cells (PBMCs) of patients with Wegeners granulomatosis (WG), relapsing polychondritis (RP) and healthy subjects (HS). DN and CD4+ Vα24+Vβ11+ NKT cells as well as CD1d‐α‐GalCer tetramer‐positive NKT cells, were significantly decreased in number in both WG and RP patients compared to those from HS. When cytokine profiles were analysed in these PBMCs upon stimulation with phorbol ester and calcium ionophore, CD4+ T cells from patients with WG and RP exhibited a Th1 bias, whereas CD4+ NKT cells from WG patients in remission showed a Th2 bias. These findings suggest that NKT cells (especially CD4+ NKT cells) play a regulatory role in Th1 autoimmunity in patients with WG and RP. The reduction in NKT cell counts appears to be associated with the low responsiveness to α‐GalCer. The dysfunction of NKT cells to recognize ligands such as α‐GalCer may also contribute to the defects observed in NKT cells from WG and RP patients.

Distribution of MEL-14+ Cells in Various Lymphoid Tissues

The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.

Donor and Recipient Specific Tolerance in Cells from Semi-Allogeneic, H-2 Subregion Compatible or Fully Allogeneic Bone Marrow Chimeras Attributable to Clonal

Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsens splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.

Sequential Analysis of the Thymocyte Differentiation in Fully Allogeneic Bone Marrow Chimera in Mice.I. Relationship between Functions and Surface Characteristics of Thymocytes

Differentiation of thymocytes according to surface phenotype, functional status and cell size was investigated using fully allogeneic bone marrow chimeras. Most of the donor-derived thymocytes obtained from chimeras 9 days after hematopoietic reconstitution were CD4-8- and IL2R+. At day 14, CD4+8+ cells became prominent in the thymus. Eighty-six per cent of thymocytes were CD4+8+ and 9% were CD4-8- at this stage. After day 21, the proportion of CD4+8- or CD4-8+ single positive cells transiently increased and then declined to normal level at day 42. Further, the mean size of CD4+ or CD8+ single positive cells in chimeric thymuses at day 21 after reconstitution was markedly larger than that at day 35. When proliferative responses to various stimuli (PMA + rIL2, anti-CD3 mAb (2C11) and anti-V beta 8 mAb (F23.1] were evaluated, significant responses were generated by thymocytes for the first time at around day 28 and the responses reached their peaks at day 35. These findings demonstrated that the process of thymocyte differentiation in the fully allogeneic chimeras was similar to ontogenic development as observed in fetal mice. However, the tempo at which the differentiation of surface phenotypes and development of functions proceeded was quite different from that seen in normal mice. The relationship among surface phenotypes, cell size and functions of developing thymocytes of bone marrow chimeras is discussed.

Fgr Expression Restricted to Subpopulation of Monocyte/Macrophage Lineage in Resting Conditions Is Induced in Various Hematopoietic Cells after Activation or Transformation

The c‐fgr gene product (Fgr) is a member of the src‐family of protein tyrosine kinases. We have established a monoclonal antibody (2H2) which recognizes the unique N‐terminal domain of the murine Fgr. In the present study, using immunohistochemical analysis and immune complex kinase assay with the 2H2, we investigated expression of Fgr in various cell populations and tissues in a murine system. In resting conditions, Fgr expression was confined to subsets of a monocyte/macrophage lineage. Thus, Fgr+ cells were detected in paracortical areas and medullas of lymph nodes, but seen only in marginal zones of the spleen and the medulla of the thymus. No Fgr+ macrophage was detected in other tissues, Peyers patches, brain, heart, lung, liver, pancreas, kidney and peritoneal cavity. However, immune complex kinase assay revealed that, upon stimulation, T and B cells as well as peritoneal macrophages expressed significant levels of Fgr molecules. Transformed cell lines of lymphoid origin, EL‐4 and LK35.2, which are T and B lineage lymphomas, respectively, also expressed Fgr molecules. Thus, various cells of hematopoietic origin appeared to possess a potentiality to express Fgr following activation or transformation. The present findings may help elucidate the functional significance of Fgr in immunologically committed cells in either activated or non‐activated conditions.

Positive and negative selection of T cell repertoires during differentiation in allogeneic bone marrow chimeras

T cells acquire immune functions during expansion and differentiation in the thymus. Mature T cells respond to peptide antigens (Ag) derived from foreign proteins when these peptide Ag are presented on the self major histocompatibility complex (MHC) molecules but not on allo-MHC. This is termed self-MHC restriction. On the other hand, T cells do not induce aggressive responses to self Ag (self-tolerance). Self-MHC restriction and self-tolerance are not genetically determined but acquired a posteriori by positive and negative selection in the thymus in harmony with the functional maturation. Allogeneic bone marrow (BM) chimera systems have been a useful strategy to elucidate mechanisms underlying positive and negative selection. In this communication, the contribution of BM chimera systems to the investigation of the world of T-ology is discussed.

Delayed Clearance of Zymosan-induced Granuloma and Depressed Phagocytosis of Macrophages with Concomitant Up-regulated Kinase Activities of Src-family in a Human Monocyte ChemoaHractant Protein-1 Transgenic Mouse

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

Mixed allogeneic chimerism with wild-type strains ameliorates atherosclerosis in apolipoprotein E-deficient mice

Atherosclerosis involves inflammatory processes between vasculartissues and hematocytes with a hyperlipidemic background. To examinewhether variations of hematocytes constitute one of the geneticcomponents in atherosclerosis, irradiated apolipoprotein E(apoE)‐deficient (apoE−/−) mice with hypercholesterolemiaand preexisting atherosclerotic lesions were reconstituted withmixed bone marrow cells (BMC) from syngeneic and wild‐type(apoE+/+; atherosclerosis‐resistant SJL or ‐susceptibleB10.S) mice. Stable mixed allogeneic chimeras with small amounts ofserum apoE were established without any detrimentalcomplications. Compared with untreated apoE−/− miceor apoE−/− mice transplanted with syngeneic BMC alone, significant reduction of the cholesterol level and significant lesionregression were observed in the mixed chimeras. Furthermore, mixedchimeras given SJL BMC showed marked reductions in numbers of lesionscompared with those reconstituted with B10.S BMC. Cholesterol levels inthe former SJL chimeras, however, were significantly higher than thosein the latter B10.S chimeras. These findings indicate that theresistance of SJL to atherosclerosis resides in the bone marrow‐derivedcells.