Shunichi Kumagai

Immunoreactive growth hormone (GH) secretion by human lymphocytes: Augmented release by exogenous GH

Peripheral blood mononuclear cells (PBMCs) from normal adults secreted small amounts of human growth hormone (GH; 0.2-0.6 pg/10(5) cells/7 days culture) as measured by a highly sensitive enzyme immunoassay. Stimulation of PBMCs with phytohemagglutinin (PHA) consistently showed a 4-6 fold increase in GH secretion. Transformed B-lymphocytes by Epstein-Barr virus also secreted GH (0.8-4.8 pg/5 x 10(4) cells/7 days culture). GH secreted by lymphocytes comigrated with pituitary GH on an Ultrogel AcA44 column. Addition of GH during the culture augmented endogenous GH secretion from PHA-stimulated PBMCs. GH-releasing hormone and a somatostatin analogue, SMS 201-995, did not affect GH secretion from non-stimulated and PHA-stimulated PBMCs. These findings suggest that both T and B lymphocytes secrete immunoreactive GH in a different manner from that in the anterior pituitary.

In vitro induction of IgG anti‐DNA antibody from high density B cells of systemic lupus erythematosus patients by an HLA DR‐restricted T cell clone

An HLA‐DR restricted T cell clone (26G11) which recognized a lymphoid cell‐derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti‐DNA antibody production in DR‐matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR‐matched normal control subjects produced large amounts of IgM anti‐DNA antibody, but did not produce IgG anti‐DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR‐matched patients with active SLE spontaneously produced a fairly large amount of IgG anti‐DNA antibody, and the production was augumented by the T cell clone. Little IgG anti‐DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD‐B) and high density B (HD‐B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti‐DNA antibody was spontaneously produced by LD‐B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti‐DNA antibody was not spontaneously produced by the HD‐B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti‐DNA antibody‐producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production.

Induction and Function of FcεRII on YT Cells; Possible Role of ADF/Thioredoxin in FcεRII Expression

Abstract The regulation of low-affinity Fc receptor for lgE (FcγRII) and the characteristics of both membrane and soluble forms of FcγRII were studied using YT cell line. We found that YT cells, a human NK like cell line, expressed FcγRII after IL-1 stimulation. Cross-linking of FceRII on IL-1-stimulated YT cells as well as the transfectant of FceRII-cDNA (YTSER) resulted in the up-regulation of IL-2Rα (p55/Tac). A 59 kDa protein phosphorylated at tyrosine residues was co-immunoprecipitated with FceRII from YTSER lysate using H107 anti-FceRII mAb. YTSER not only expressed FceRII on their surface but also secreted soluble form of FceRII (sFceRII/sCD23; IgE binding factor). Affinity purification revealed that sFceRII released from YTSER is heterogeneous and consisted of several proteins differing in molecular weight. Both EBV + B cells and HTLV-1+ T cells are high producers of ATL derived factor (ADF)/ thioredoxin (TRX) and express FceRII and IL-2Rα respectively. To clarify the mechanism of FceRII and IL-2Ra induction by ADF/TRX, we examined the effect of ADF/TRX on the bindability of nuclear factor κB (NF-κB), which is known to regulate IL-2Rα gene expression. In the gel shift assay, ADF/TRX was shown to enhance the bindability of NF-κB to its responsive element.

Elevated serum levels of IgE-binding factor/soluble CD23 in bullous pemphigoid

IgE-related abnormalities and impaired B cell function have been reported in patients with bullous pemphigoid (BP). CD23 is a low affinity Fc epsilon RII on haematopoietic cells and its soluble form (sCD23) is involved in B cell growth and differentiation. In order to determine the role of CD23 in the pathomechanisms of BP, the present study assayed the level of sCD23 in sera of BP patients using an ELISA method. Levels of sCD23 were elevated in BP patients compared with mean levels in the sera of normal control. Furthermore, the increase in sCD23 levels correlated with the degree of disease activity. A significant correlation was found between sCD23 and serum IgE levels in BP sera, but not in normal control sera. These results suggest that sCD23 is an important index for monitoring BP with respect to IgE-related abnormalities and impaired B cell function.

A human T cell line established from a patient with Sézary syndrome: application for assay of human interleukin 2 (IL-2)

A human T cell line, designated Sez 627, was established from a patient with Sézary syndrome. These clonal T cells have been cultured for more than 2 years in the presence of IL-2 without any antigen stimulation. The surface phenotype of Sez 627 was OKT3+, OKT4-, OKT8+, and Tac+, and infection with human T lymphotrophic virus type I (HTLV-I) was demonstrated by Southern blot hybridization analysis. In human IL-2 assay, Sez 627 cells were found to be superior to murine CTLL-2 cells with respect to their unresponsiveness to phorbol 12-myristate 13-acetate (PMA), and species specificity for human IL-2, as well as better cryopreservation, although they were less sensitive to IL-2 than CTLL-2 cells. Using Sez 627 cells, IL-2 production by lymphocytes from patients with systemic lupus erythematosus (SLE) was examined. Decreased IL-2 production was observed in the patients with active SLE but not in most patients with inactive SLE. These findings suggest that Sez 627 is a useful human T cell line for human IL-2 assay.

Immune Regulatory Abnormalities in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by B-cell hyperactivity, autoantibody formation, and by antibody- mediated tissue damage which leads to tissue inflammation in many organs. Genetic factors have been shown to play a role in the disorder on the basis of both family studies and associations with particular immuneassociated antigens coded for in the major histocompatibility complex (Benacerraf and McDevitt, 1972; Decker et al., 1979). However, multiple genes and multiple factors appear to modify the expression of illness (see Table 1). Moreover, the multiplicity of immune defects in patients with SLE and the differences among patients have complicated establishment of abnormalities which underlie the illness and those which result therefrom.

Enhanced production of transforming growth factor‐beta (TGF‐β) during autologous mixed lymphocyte reaction of systemic sclerosis patients

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF‐β is suggested to be related to skin fibrosis, we examined the production of TGF‐β from peripheral mononuclear cells (MNC) of SSc patients. Since anti‐TGF‐β neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF‐β was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF‐β in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF‐β excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR‐like in vivo responses.

The presence of antibodies to purified p24gag protein of HTLV-I in sera of patients with systemic lupus erythematosus (SLE)

Sera of patients with systemic lupus erythematosus (SLE) were tested for their reactivity to HTLV-I by western blotting (WB). Seven (18%) of 40 SLE serum samples reacted to the p24gag protein of HTLV-I by WB using purified gag antigens. The specificity of anti-p24gag antibodies in the SLE sera was confirmed by competitive inhibition on WB. Two of the seven patients were shown to be HTLV-I carriers, because HTLV-I infected T cell lines were easily established from their peripheral blood mononuclear cells (PBMC). Except for these two carrier patients, the gag proteins were not detected in the lysates of PBMC by WB using anti-p24gag and anti-p19gag monoclonal antibodies. The gag and pX genes of HTLV-I were not detected by PCR in PBMC of the SLE patients, with the exception of the 2 HTLV-I carrier patients. These results show no direct involvement of HTLV-I in the etiology of SLE. However, the existence of a specific antibody to p24gag in the sera of some of the noncarrier SLE patients suggests a crossreactivity to either unknown viruses or some autoantigens.

Sensitive Sandwich Enzyme Immunoassay of Human Interleukin-2 Produced In Vitro by Peripheral Blood Mononuclear Cells

Abstract A sensitive sandwich enzyme immunoassay for human inter-leukin-2 (hIL-2) using monoclonal antibody IS described. A monoclonai anti-hIL-2 IgG-coated poiystyrene ball was incubated with hIL-2 and subsequently with affinity-purified rabbit anti-hIL-2 Fab-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. There was no cross-reaction with interleukins 1α and 1bT, epidermal growth factor, insulin and other protein hormones. The detection limit of hIL-2 was 3 pg/tube or 30 ng/1 using 0.1 ml of cuiture supernatant. When peripheral blood mononuclear cells from healthy subjects aged 22-62 yr were cultured in the absence and presence of phytohemagglutinin P for 48 h, hIL-2 levels in the culture supernatants were <0.03-0.10 μg/1 and 0.18-3.7 μg/1, respectively.

Qualitative difference of anti‐DNA antibody‐producing cell precursors in the pre‐immune B cell repertoire between normal and lupus‐prone mice

The precursor frequency for anti‐DNA antibody‐producing cells in the pre‐immune B cell repertoire was investigated in young female BALB/c and NZW mice, and in young and aged female NZB ± NZWFl (B/WF1) mice. Spleen cells from these mice were diluted serially and stimulated polyclonally in vitro with lipopolysaccharide (LPS) and IL‐4 to induce both IgM and IgG1 production. The results demonstrated that there existed virtually no difference in precursor frequency for IgM anti‐DNA antibody‐producing cells between normal and lupus mice, confirming previous observations made by other investigators. In contrast, the number of precursors for IgG 1 anti‐DNA antibody‐producing cells was much higher in young and old B/WF1 mice than in normal mice. These results suggest that the high frequency of precursors for IgG1 anti‐DNA antibody‐producing cells in the pre‐immune B cell repertoire of B/WF1 mice is a crucial factor for the pathogenesis of systemic lupus erythematosus.