Kimiko Kasahara

Suppression of immunoglobulin production of lymphocytes by intravenous immunoglobulin

The proliferative responses and the immunoglobulin production of peripheral blood mononuclear cells to pokeweed mitogen were dose-dependently suppressed by sulfonated intravenous immunoglobulin (IVIG), polyethylene glycol-treated IVIG, pH 4-treated IVIG, or human γ-globulin, but they were not or only slightly suppressed by human serum albumin or pepsin-treated IVIG. Moreover, the suppression of immunoglobulin production by sulfonated IVIG, polyethylene glycol-treated IVIG, or pH 4-treated IVIG was seen in the cases in which B cells preincubated with IVIGs were cocultured with T cells and monocytes preincubated with or without IVIGs and in the cases in which monocytes preincubated with IVIGs were cocultured with T cells and B cells preincubated with or without IVIGs. However, in the cases in which only T cells were preincubated with IVIGs, immunoglobulin production was not suppressed. The suppression of the monocyte function by IVIGs tended to be less than the suppression of the B-cell function by IVIGs. Moreover, the suppression by IVIGs was blocked by antihuman IgG Fc. Our results suggest that IVIGs suppress the immunoglobulin production of lymphocytes through suppression of the B-cell function and the antigen presenting-cell function by attachment of IVIGs to Fc receptors of B-cell membranes and antigen presenting-cell membranes.

Reduced IFNγ production in response to IL‐12 stimulation and/or reduced IL‐12 production in atopic patients

Several studies have shown that interleukin (IL)‐4 and interferon‐gamma (IFNγ) are important for the regulation of IgE production and that IL‐12 induces IFNγ.

Intravenous Immunoglobulins Suppress Immunoglobulin Productions by Suppressing Ca2+ ‐Dependent Signal Transduction Through Fc γ Receptors in B Lymphocytes

A high dose intravenous immunoglobulin (IVIG) therapy is used in the treatment of a wide range of autoimmune disorders. However, the mechanisms of the action of IVIGs remain poorly understood. To analyse the mechanisms of effects of IVIGs on immunoglobulin (Ig) production of B cells, the effects of IVIGs on B lymphoblastoid cell lines transformed by Epstein‐Barr virus (LCLs) were investigated. The productions of IgG or IgM of LCLs were dose‐dependently suppressed by polyethylene glycol (PEG)‐treated IVIG or pH 4‐treated 1VIG though the productions were not or only slightly suppressed by pepsin‐treated IVIG. The suppression by IVIGs was blocked by anti‐human IgG Fc or anti‐Fc γ RII. C μ gene expression and μ s C terminal gene expression of LCLs were suppressed by PEG‐treated IVIG, whereas neither C μ gene expression nor μ s C terminal gene expression of LCLs were suppressed by pepsin‐treated IVIG. Although the increase in intracellular calcium concentration in LCLs was not suppressed by pepsin‐treated IVIG, the increase was suppressed by PEG‐treated IVIG. This suppressing effect of PEG‐treated IVIG on intracellular calcium concentration of LCLs was blocked by anti‐human IgG Fc or anti‐ Fc γ RII. Our results suggest that IVIGs suppressed the Ca2+ ‐dependent signal transduction through Fc γ R on B‐cell membrane, consequently, the transcription of C γ mRNA, especially secreted γ mRNA was suppressed in the B cells.

Molecular analysis of B-cell differentiation in selective or partial IgA deficiency

Selective IgA deficiency is the most common form of primary immunodeficiency, the molecular basis of which is unknown. To investigate the cause of selective IgA deficiency, we examined what stage of B‐cell differentiation was blocked. DNA and RNA were extracted from three Japanese patients with selective IgA deficiency and three with a partial IgA deficiency. In selective IgA deficiency patients, Iα germline transcript expression levels decreased and α circle transcripts were not detected. Stimulation with PMA and TGF‐β1 up‐regulated Iα germline and α circle transcripts. In some patients, IgA secretion was induced by stimulation with anti‐CD40, IL‐4 and IL‐10. In partial IgA deficiency patients, Iα germline, α circle transcripts and Cα mature transcripts were detected in the absence of stimulation. Our findings suggest that the decreased expression level of Iα germline transcripts before a class switch might be critical for the pathogenesis of some patients with selective IgA deficiency. However, in patients with a partial IgA deficiency, B‐cell differentiation might be disturbed after a class switch.

Reduced secreted μ mRNA synthesis in selective IgM deficiency of Bloom's syndrome

Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Blooms syndrome. Although the percentages of surface Ig‐earing cells were not reduced, the numbers of Ig‐ecreting cells were markedly reduced. The membran‐ound μ(μm) and secreted μ (μs) mRN As arc produced from transcripts of a single immunoglobulin μ gene by alternative RNA processing pathways. The control of μs mRNA synthesis depends on the addition of poly(A) to μs ‐erminal segment. In both patients, μ mRNA was well detected but μs ‐erminal mRNA was scarcely detected, suggesting that μ mRNA was well transcribed but μs mRNA was not. There was, at least, no mutation or deletion in the μs ‐erminal coding sequence, the RNA splice site (GG/TAAAC) at the 5’ end of μs ‐erminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Blooms syndrome is due to an abnormality in the maturation of surface Ig‐earing B cells into Ig‐ecreting cells and a failure of μs mRNA synthesis. Moreover, reduced μs mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the μ gene.

Defective Calcium-Dependent Signal Transduction in T Lymphocytes of Ataxia-Telangiectasia

T‐cell functions of two patients with ataxia‐telangiectasia were investigated. Patients with ataxia‐telangiectasia had reduced percentages of circulating CD3+ cells and CD4+ cells, although neither patient had a reduced percentage of circulating CD8+ cells. The proliferative responses and interleukin‐2 production of peripheral blood mononuclear cells to T‐cell mitogens were reduced in the patients. The intracellular calcium concentration in T cells or CD4+ cells from both patients was only slightly increased after phytohaemagglutinin stimulation. Moreover, the concentration after OKT3 stimulation was not or only slightly increased in T cells or CD4+ cells from both patients. Our results suggest that the functional defect of T cells is caused by defective Ca2+‐dependent signal transduction through the CD3 complex of the surface in T cells of ataxia‐telangiectasia.

Age-related changes in intracellular cytokine profiles and Th2 dominance in allergic children

The unbalanced T helper response has been pointed out in allergic diseases. Especially in childhood, it is important to consider the development of acquired immunity. We investigated the relationship between age and Th1, Th2, Tc1 or Tc2 cells. In addition, Th1, Th2, Tc1 or Tc2 cells in allergic diseases were compared with control subjects. Thirty‐four healthy controls (0–40 years old), 200 samples of cord blood, nine patients with atopic dermatitis (AD) (1–3 years old) and five patients with bronchial asthma (BA) (2–6 years old) were studied. Surface staining with CD4, CD8 and intracellular staining with anti‐interferon‐γ (IFN‐γ) and anti‐interleukin (IL)‐4 were carried out, and analyzed by using flow cytometry. In the healthy controls, the percentages of Th1, Tc1 or Th2 showed positive correlation with age. The absolute numbers of Th1 or Tc1 also correlated with age. Cord blood with a family history of allergic disease showed no significant difference compared to that without a family history. The percentage of Th2 in AD and BA patients was significantly higher than in the age‐matched healthy controls. The increase in Th1, Th2 and Tc1 with age might reflect on the development of acquired immunity. Age matching is important when evaluating the cytokine profiles of T cells. In allergic diseases, although cord blood showed a Th1‐dominant pattern, it changed to Th2 dominance in childhood, and this may reflect on some genetic background.

Hyper-IgM syndrome with putative dominant negative mutation in activation-induced cytidine deaminase

BACKGROUND Hyper-IgM immunodeficiency is an immunologic disorder characterized by normal or increased serum IgM levels and reduced serum IgG and IgA levels caused by the disruption of Ig class switching in B cells. The gene encoding activation-induced cytidine deaminase (AID) is responsible for the autosomal recessive form of hyper-IgM syndrome. OBJECTIVE To investigate the relationship between the AID gene mutation and the clinical phenotype, we analyzed the AID gene in a female Japanese patient with the autosomal recessive form of hyper-IgM syndrome. METHODS Genomic DNA and cDNA were extracted from neutrophils and analyzed by means of PCR. The AID gene was expressed as a glutathione S-transferase fusion protein. RT-PCR was performed after stimulation of the patients PBMCs with phorbol myristate acetate and TGF-beta. RESULTS Despite significantly low serum IgG levels, our patient had not shown a predisposition to any severe infections, even without Ig replacement therapy. We identified a point mutation resulting in the stop codon in exon 5 of the AID gene (R190X) in the patient. No other mutations of the AID gene were detected in the patient. The same mutation was not detected in other members of her family. The mutant allele fused with the glutathione S-transferase protein was expressed stably at the same level as the normal allele. The AID gene expression in the patient was induced by phorbol myristate acetate and TGF-beta. CONCLUSION The mutation of the AID gene is assumed to be of the dominant negative form. This is the first report of a dominant negative form of the mutation in vivo.

IFN-gamma production in response to IL-18 or IL-12 stimulation by peripheral blood mononuclear cells of atopic patients.

Background Several studies have shown that interleukin (IL)‐4 and interferon‐gamma (IFN‐γ) are important for the regulation of immunoglobulin E (IgE) production and that IL‐18 and IL‐12 induce IFN‐γ.

Reduced Expression of the Interferon‐Gamma Messenger RNA in IgG2 Deficiency

The specific defect that causes IgG2 deficiency, which is one of the primary immunodeficiencies, is unknown. Recently, it was shown that interferon‐γ (IFN‐γ) induces synthesis of human germline Cγ2 transcripts. In the authors’ previous study and the present one, peripheral blood lymphocytes (PBLs) of all five tested patients with IgG2 deficiency failed to produce enough IFN‐γ when stimulated with phytohaemagglutinin or concanavalin A although they produced a sufficient amount of interleukin‐2 (IL‐2). The low level of IgG2 production in pokeweed mitogen‐stimulated PBLs of four tested patients was improved by the addition of recombinant IFN‐γ. In this study, the amount of IFN‐γ messenger RNA showed various degrees of reduction in all five tested patients. Sequence analysis of the IFN‐γ coding regions and flanking regions revealed neither a point mutation nor a deletion for any of the patients. Thus the results suggest that the reduced expression of IFN‐γ messenger RNA may play an important role in the IgG2 deficiency of these patients.