Soichi Haraguchi

Pyogenic bacterial infections in humans with IRAK-4 deficiency.

MyD88 is a key downstream adapter for most Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 deficiency in mice leads to susceptibility to a broad range of pathogens in experimental settings of infection. We describe a distinct situation in a natural setting of human infection. Nine children with autosomal recessive MyD88 deficiency suffered from life-threatening, often recurrent pyogenic bacterial infections, including invasive pneumococcal disease. However, these patients were otherwise healthy, with normal resistance to other microbes. Their clinical status improved with age, but not due to any cellular leakiness in MyD88 deficiency. The MyD88-dependent TLRs and IL-1Rs are therefore essential for protective immunity to a small number of pyogenic bacteria, but redundant for host defense to most natural infections.

IRAK-4- and MyD88-Dependent Pathways Are Essential for the Removal of Developing Autoreactive B Cells in Humans

Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for interleukin-1 receptor-associated kinase 4 (IRAK-4), myeloid differentiation factor 88 (MyD88), and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal, and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8, and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive mature naive B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88-, and UNC-93B-deficient patients did not display autoreactive antibodies in their serum or develop autoimmune diseases, suggesting that IRAK-4, MyD88, and UNC-93B pathway blockade may thwart autoimmunity in humans.

Immunosuppressive retroviral peptides: cAMP and cytokine patterns

The mechanism(s) by which retroviral proteins exert immunosuppressive influences has remained enigmatic. Here, Soichi Haraguchi, Robert Good and Noorbibi Day propose that induction of intracellular cAMP by a synthetic, immunosuppressive, retroviral envelope peptide causes a shift in the cytokine balance, leading to suppression of cell-mediated immunity by upregulation of interleukin 10 (IL-10) and downregulation of IL-2, IL-12 and tumor necrosis factor alpha production. This may be a crucial step towards generation of immune dysfunction.

Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections.

Studies of the effects of retroviruses on the immune system, which date back through thirty years of investigations, are reviewed. In the earliest published studies in the 1960s, it was demonstrated that mice infected with oncogenic viruses were immunosuppressed. Since then, numerous articles have been published describing profound immunodeficiencies observed in vivo in humans infected with human immunodeficiency virus and in animals such as cats infected with the feline immunodeficiency virus. In vitro investigations have shown that inactivated retroviruses or transmembrane envelope protein pl5E as well as a synthetic 17‐amino acid peptide (CKS‐17) impressively conserved within the transmembrane envelope protein of several animal or human retroviruses are highly immunosuppressive. More recently, dysfunction of cytokines produced by CKS‐17 at both a cellular and molecular level have been found to mimic influences observed in vivo in patients infected with the human immunodeficiency virus. CKS‐17 has also been shown to induce cAMP in vitro. The significance of these observations to understanding the immunological disturbances observed in malignancy, cytokine biosynthesis, and modulations of immune functions through cAMP is discussed. J. Leukoc. Biol. 61:654–666; 1997.

A potent immunosuppressive retroviral peptide: cytokine patterns and signaling pathways

A synthetic bioactive peptide composed of 17 amino acids (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane envelope proteins induces not only significant immunoregulatory functions but also exhibits Th1-inhibiting properties, as described by its ability to suppress cell-mediated immunity and inhibit the production of interleukin (IL) 12, IL-2, gamma interferon, and tumor necrosis factor alpha, while enhancing IL-10. An important molecular mechanism responsible for the observed cytokine profiles by CKS-17 is provided by our findings demonstrating that this small peptide activates several intracellular signaling molecules, i.e., elevates intracellular cyclic adenosine monophosphate (cAMP) levels, and induces phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, mitogen-activated protein kinase/ERK kinase (MEK), protein kinase D, Raf1, and phospholipase C gamma1 (PLCγ1). The activation of ERK1/2 is via the PLCγ1-protein kinase C-Raf1-MEK signaling cascade. The activation of both ERK1/2 and cAMP appears to be via a mechanism sensitive to AG879, a receptor tyrosine kinase inhibitor, but not to AG825, AG1296, or AG1478. Furthermore, phosphoinositide-3 kinase appears to mediate the CKS-17-induced activation of ERK1/2, but not of cAMP. A specific amino acid sequence as well as the dimerization of this peptide is required to confer these biological activities. The results obtained are compelling and reproducible. This highly conserved molecule may enable us to understand a basic mechanism(s) of intracellular signaling pathways, regulation of Th1/Th2 cytokines, immunosuppression, and immunologic tolerance.

HIV-1 RECOMBINANT GP41 INDUCES IL-10 EXPRESSION AND PRODUCTION IN PERIPHERAL BLOOD MONOCYTES BUT NOT IN T-LYMPHOCYTES

The effects of recombinant gp41 (rgp41) protein of the human immunodeficiency virus type 1 (HIV-1) on interleukin 10 (IL-10) expression and production using human peripheral blood mononuclear cells was investigated. Expression of IL-10 mRNA was demonstrated within 3 h of cell exposure to endotoxin-free rgp41 by RT-PCR and Northern blot analyses in a time- and dose-dependent manner. IL-10 protein was detected in the supernatants of peripheral blood mononuclear cells following stimulation with rgp41 also in a dose dependent manner. Fractionation of peripheral blood mononuclear cells showed that purified monocytes but not purified T-lymphocytes induced expression of IL-10 mRNA by rgp41. Recombinant HIV-1 gp120 exhibits similar influences on the induction of IL-10. These results indicate that both of these components of envelope proteins may play an important role in HIV related immunomodulation by influencing regulatory functions of monocytes and macrophages.

A Retroviral-Derived Immunosuppressive Peptide Activates Mitogen-Activated Protein Kinases

The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.

Cytokine mRNA Profile in Lipoid Nephrosis: Evidence for Increased IL-8 mRNA Stability

Background/Aims: Proteinuria in idiopathic minimal lesion nephrotic syndrome (IMLNS) is presumed to be due to the effect of circulating factors on glomerular permeability to plasma proteins. This study examines the expression of messenger ribonucleic acid (mRNA) for cytokines thought to mediate glomerular inflammation during different stages of the nephrotic syndrome. Methods: Messenger RNA expression and stability from peripheral blood mononuclear cells of IMLNS patients in relapse and in remission, and age matched normal controls were measured using a ribonuclease protection assay (RPA). The spontaneous and Interleukin 2 (IL-2) stimulated mRNA expression were studied. Results: Spontaneous mRNA expression for Interleukin 8 (IL-8) from IMLNS patients in relapse was significantly increased when compared to IMLNS patients in remission and normal controls (p < 0.05). After 14 h of IL-2 stimulation, mRNA IL-8 levels expressed by IMLNS PBMC patients in remission were not different from those observed in normal controls. However, after 5 days of PBMC incubation, a significant increase in mRNA for IL-8 in IMLNS patients compared to controls was found (p < 0.01). Stability assay demonstrated that IL-8 mRNA transcript from the nephrotic patients remained higher than those from controls and showed a significantly prolonged life t1/2 (p = 0.02). Conclusions: IL-8 mRNA expression is increased in IMLNS patients in relapse. Moreover, stability studies show that IL-8 mRNA life t1/2 is prolonged due to altered post-transcriptional regulation. This finding may explain the elevated serum IL-8 levels observed in these patients during relapse and may have pathogenic significance since IL-8 has been shown to induce proteinuria in the experimental animal.

Suppression of human interferon-γ production by a 17 amino acid peptide homologous to the transmembrane envelope protein of retroviruses: Evidence for a primary role played by monocytes

CKS-17, a synthetic amino acid peptide homologous to a highly conserved region of retroviral transmembrane protein exerts a suppressive action on staphylococcal enterotoxin A (SEA)-induced the production of IFN-gamma by human peripheral blood mononuclear cells (PBMC) (Ogasawara et al., J. Immunol. 141, 615, 1988). This action has been shown in the present study to be preceded by dramatic clustering of PBMC. Clusters appear within 3 hr of exposure of PBMC to CKS-17; they are dose dependent, inhibited by cycloheximide, and require a temperature of 37 degrees C. The cells in the clusters are predominantly monocytes. Although it has been previously shown that CKS-17 inhibits monocyte-mediated killing by inactivating IL-1 (Kleinerman et al., J. Immunol. 139, 2329, 1987) and production of IL-2 by murine thymoma cells treated with IL-1 (Gottlieb et al., J. Immunol. 142, 4321, 1989), in the present study we show that IL-1 does not prevent clustering of PBMC by CKS-17. Using CKS-17 and highly purified monocytes or lymphocytes, profound alterations occur only with monocytes, as revealed by light or electron microscopy. SEA- or staphylococcal enterotoxin B-induced production of IFN-gamma is inhibited when highly purified monocytes pretreated with CKS-17 are cocultured with highly purified T lymphocytes. Thus, CKS-17 induces dramatic clustering of cells apparently by inducing alterations of monocytes but not lymphocytes, suggesting that CKS-17 may interfere with the capacity of monocytes to facilitate production of IFN-gamma by T lymphocytes.

Inherited mannose-binding lectin deficiency as evidenced by genetic and immunologic analyses: association with severe recurrent infections

BACKGROUND Mannose-binding lectin (MBL), an acute-phase serum protein of hepatic origin, plays an essential protective role in host innate immunity in targeting microbial pathogens for destruction via opsonization, enhancement of phagocytosis and complement activation. MBL deficiency, characterized by low serum MBL, has been attributed to genetic mutations in both structural and promoter regions of the gene coding for the protein. Concomitant MBL deficiency in patients with chronic immunologic disease has been associated with increased susceptibility to complicating infections that may hasten disease progression. OBJECTIVE Few cases of inherited MBL deficiency in adults and possible associations with recurrent infection have been reported. To address this issue, we investigated the MBL profile of four generations within a single adult family whose members have experienced a variety of persistent infections. METHODS MBL serum levels and MBL genotypes of each participating family member were ascertained by enzyme-linked immunoadsorbent assay and reverse transcriptase-polymerase chain reaction, respectively. MBL complement activation, as measured by C4b deposition against mannan-coated wells, was assayed using an enzyme-linked immunoadsorbent assay. Routine immunologic and cellular tests were carried out to evaluate the immunologic status of each family member. RESULTS Six of the 7 family members screened carried one or more of the variant MBL alleles in their genotype and had correspondingly low serum MBL and reduced ability to affect C4b opsonization. Medical histories of the participating family members revealed an array of mild to severe recurrent infections despite no apparent immunodeficiency. CONCLUSIONS Our studies show that MBL deficiency is an inherited characteristic and may be a crucial factor in maintaining immunologic health.