IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.

[Recent advances in tuberculosis immunity].

Primary tuberculosis infection is acquired by the inhalation of droplets containing Mycobacterium tuberculosis (MTB) bacilli. Only 5-10% of those individuals infected by MTB develop clinical diseases, and disease presentation itself is heterogeneous, suggesting that host factors play a large role in disease susceptibility. Protective immunity in the lung against MTB consist of the innate immunity in which alveolar macrophages play an central role, and the acquired immunity including various type of effector T cells. Recent studies show that the important roles of the receptors which recognize MTB for the development of protective immunity, the difference in the anti-MTB activity of macrophages between human and mice, the macrophage-heterogeneity that affects the anti-MTB activity, the role of IL-10 in the activation of anti-MTB activity of human macrophages, and the role of Th17/IL-17, Th22/ IL-22 and TNF in the protective immunity against human tuberculosis. In this review, these recent advances in tuberculosis immunity will be described.

Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis.

Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-alpha, and IL-6, than typical intestinal resident macrophages (CD14-CD33+ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-alpha compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14+ macrophages contributed to IFN-gamma production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-alpha. Furthermore, the IFN-gamma produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-gamma-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.

Novel 12-membered non-antibiotic macrolides from erythromycin A; EM900 series as novel leads for anti-inflammatory and/or immunomodulatory agents.

Herein, we report the design and synthesis of the novel 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900), which was found to be a potent anti-inflammatory and/or immunomodulatory agent, capable of promoting monocyte to macrophage differentiation. This molecule shows improved acid stability, does not exhibit any anti-bacterial activity and has relatively low cytotoxicity against THP-1 cells. In addition, one of its analogues, (8R,9S)-4″,13-O-diacetyl-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM911), was found to be twice as effective as EM900.

Erythromycin derivatives inhibit HIV-1 replication in macrophages through modulation of MAPK activity to induce small isoforms of C/EBPbeta.

Macrophages (MPhis) are a major source of HIV-1 especially in patients with tuberculosis. There are MPhis that are permissive and those that restrict HIV-1. Regulation of hematopoietic cell kinase (Hck) activity and selective expression of CCAAT enhancer binding protein beta (C/EBPbeta) isoforms greatly contribute to determine distinct susceptibility of MPhis to HIV-1. Resistance is attributable to reduced expression of Hck and augmented expression of an inhibitory small isoform of C/EBPbeta. Derivatives of erythromycin A (EMA) EM201 and EM703 inhibit the replication of HIV-1 in tissue MPhis, at posttranscriptional and translational levels. We demonstrate that EM201 and EM703 convert tissue MPhis from HIV-1 susceptible to HIV-1 resistant through down-regulation of Hck and induction of small isoforms of C/EBPbeta. These drugs inhibit p38MAPK activation which is expressed only in susceptible tissue MPhis. Activated CD4(+)T cells stimulate the viral replication in HIV-1 resistant MPhis through down-regulation of small isoforms of C/EBPbeta via activation of ERK1/2. EM201 and EM703 can inhibit the MAPK activation and inhibit the burst of viral replication produced when CD4(+)T cells and MPhis interact. These EM derivatives may be highly beneficial for repression of residual HIV-1 in the lymphoreticular system of HIV-1-infected patients and offer great promise for the creation of new anti-HIV drugs for the future treatment of AIDS patients.

Azithromycin, a 15-membered macrolide antibiotic, inhibits influenza A(H1N1)pdm09 virus infection by interfering with virus internalization process.

The pandemic influenza 2009 (A(H1N1)pdm09) virus currently causes seasonal and annual epidemic outbreaks. The widespread use of anti-influenza drugs such as neuraminidase and matrix protein 2 (M2) channel inhibitors has resulted in the emergence of drug-resistant influenza viruses. In this study, we aimed to determine the anti-influenza A(H1N1)pdm09 virus activity of azithromycin, a re-positioned macrolide antibiotic with potential as a new anti-influenza candidate, and to elucidate its underlying mechanisms of action. We performed in vitro and in vivo studies to address this. Our in vitro approaches indicated that progeny virus replication was remarkably inhibited by treating viruses with azithromycin before infection; however, azithromycin administration after infection did not affect this process. We next investigated the steps inhibited by azithromycin during virus invasion. Azithromycin did not affect attachment of viruses onto the cell surface, but blocked internalization into host cells during the early phase of infection. We further demonstrated that azithromycin targeted newly budded progeny virus from the host cells and inactivated their endocytic activity. This unique inhibitory mechanism has not been observed for other anti-influenza drugs, indicating the potential activity of azithromycin before and after influenza virus infection. Considering these in vitro observations, we administered azithromycin intranasally to mice infected with A(H1N1)pdm09 virus. Single intranasal azithromycin treatment successfully reduced viral load in the lungs and relieved hypothermia, which was induced by infection. Our findings indicate the possibility that azithromycin could be an effective macrolide for the treatment of human influenza.

Non-antibiotic 12-membered macrolides: design, synthesis and biological evaluation in a cigarette-smoking model.

The 14-membered macrolide erythromycin A expresses three distinct biological properties, including antibacterial activity, gastrointestinal motor-stimulating activity and anti-inflammatory and/or immunomodulatory effects. Although low-dose, long-term therapy using 14- and 15-membered macrolides displaying anti-inflammatory and/or immunomodulatory activity effectively treats diffuse panbronchiolitis and chronic sinusitis, bacterial resistance may emerge. To address this issue, we developed the 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900) that promotes monocyte to macrophage differentiation, a marker for anti-inflammatory and/or immunomodulatory effects, without possessing antibacterial activity. In this article, we report that the new macrolide derivative (8R,9S) -de(3'-N-methyl)-3'-N-(p-chlorobenzyl)-de(3-O-cladinosyl)-3-dehydro-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A 12,13-carbonate (EM939) exhibited stronger promotive activity for monocyte to macrophage differentiation than that of the parent compound EM900 in addition to reduced cytotoxicity toward THP-1 cells and antibacterial inactivity. In a cigarette-smoking model used to simulate chronic obstructive pulmonary disease (COPD), the EM900 derivatives significantly attenuated lung and alveolar inflations, functionally and histologically, via oral administration. Because of these marked therapeutic effects, non-antibiotic EM900 derivatives may become central to the treatment of chronic inflammatory diseases such as COPD.

Eicosapentaenoic acid inhibits CSF-induced human monocyte survival and maturation into macrophage through the stimulation of H2O2 production.

Human studies suggest a beneficial effect of eicosapentaenoic acid (EPA)-supplemented diets on atherosclerotic and atherothrombotic disorders as well as autoimmune and inflammatory diseases and tumors. The effects of EPA on human monocyte survival and maturation into macrophage are not yet known. We studied the effects of EPA on the survival and development into macrophage of human monocyte treated with colony-stimulating factor (CSF). We have found that EPA induces cell death of the monocyte via apoptosis, even in the presence of M-CSF or GM-CSF, and inhibits differentiation from the monocyte to macrophage by inducing H2O2 production. In contrast to the effect of EPA on monocytes, EPA did not induce cell death of monocyte-derived macrophages. Such an apoptosis inducing effect on monocytes by EPA may contribute to the efficacy of EPA in atherosclerosis and autoimmune diseases.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.

Macrophages have various functions and play a critical role in host defense and the maintenance of homeostasis. However, macrophages are heterogeneous and exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression, and function. When blood monocytes are cultured in medium alone in vitro, monocytes die, and colony-stimulating factors (CSFs) such as macrophage (M)-CSF or granulocyte-macrophage (GM)-CSF are necessary for their survival and differentiation into macrophages. However, M-CSF-induced monocyte-derived macrophages (M-Mphi) and GM-CSF-induced monocyte-derived macrophages (GM-Mphi) are distinct in their morphology, cell surface antigen expression, and functions, including Fcgamma receptor mediated-phagocytosis, H2O2 production, H2O2 sensitivity, catalase activity, susceptibility to human immunodeficiency virus type 1 and Mycobacterium tuberculosis, and suppressor activity. The characteristics of GM-Mphi resemble those of human alveolar macrophages.

[Up-to-date understanding of tuberculosis immunity].

This symposium was organized to provide the up-to-date knowledge on tuberculosis immunity, especially on the understanding of cytokines or Th1 cells involved in pathophysiology/protective immunity and vaccine development. Dr. Kazuo Kobayashi (Osaka City Univ.) reported their findings on the immune response to bioactive lipid component from M. tuberculosis, trehalose-dimycolate (TDM) and sulfolipid (SL) in mice. Their unique and novel finding was that TDM is capable of inducing T-dependent immune response in euthymic mice. The specific immune response in TDM-immune mice was consisting of CD4+ cell response and expression of chemokines, inflammatory cytokines and then TH1-related cytokines. In contrast, SL did not show such an activity. TDM may be one of the protective antigens and may modulate the specific immune response of the host. Dr. Isamu Sugawara's group (JATA) has examined the involvement of various cytokines in the host response to aerosolic infection with virulent strain of M. tuberculosis by using cytokine-knockout mice. The single deletion of IFN-gamma or TNF alpha resulted in a severe lesion of multiple necrosis without granuloma, and cytokine mRNA level other than knocked out cytokine was normal, suggesting that IFN-gamma and TNF alpha are principally important cytokines. In knockout mice for IL-12 or IL-18, necrotic lesion was not induced after infection and the pathological change was not so significant as in IFN-gamma/TNF alpha knockout mice. By using IFN-gamma knockout mice, it became possible to generate a granulomatous lesion with central necrosis and cavity resembling the lesion in humans. These mouse model appeared to be useful in the analysis of pathophysiology of human tuberculosis. Dr. Kazuyoshi Kawakami (Ryukyu Univ.) reported the importance of TH1 cytokines in anti-tuberculous immunity. By using IL-12, IL-18 knockout mice or double knockout mice, it was shown that IL-12 exhibits more important role than IL-18 in the protection. A possible contribution of IL-23 was also suggested. In most of the clinical cases of tuberculosis, the production of IL-12, IL-18 and IFN-gamma is increased, however, the group of relatively lower cytokine production did not respond well to the treatment. In addition, the plasma level of one of the chemokines, IP-10, was shown to be an indicator for the severity of the disease. Thus, some cytokines appear to be employable for the novel treatment in the near future. Dr. Saburo Yamamoto (NIH) summarized the recent advance in the understanding of biological function of CpG motifs. Immunostimulatory DNA is effective in the modulation of TH1/TH2 polarity and the enhancement of protective immunity to M. tuberulosis in animals. CpG motif (immunostimulatory DNA) appears to exert its activity by signaling cascade via TLR9 resulting in NF-kappa B activation and cytokine gene expression. Analysis of basic mechanism of action by CpG motif should pave the way to the clinical application in the future. Dr. Masaji Okada (Kinki Chuo Hospital) reported the current situation in the development of novel vaccines against tuberculosis. They have extensively constructed and examined the efficacy of various types of vaccines including subunit, DNA and recombinant BCG vaccines. Various vector systems have been tested for DNA vaccine. As immunizing antigens, a-Ag, ESAT-6, HSP65, 38kD-lipoprotein and so on have been employed. A large body of experimental data are accumulating for final evaluation, and among them, it is noteworthy to mention that HSP65DNA + IL-12DNA was 100 times more effective than conventional BCG in animal model. Among subunit vaccines, Mtb72f vaccine appears to be one of the promising candidates. In addition to the trial with various candidates, they have established a new mouse model, SCID/human PBL. This model animal has been employed for the development of vaccine effective for the induction of ESAT-6-specific human T cells.

Leucomycin A3, a 16-membered macrolide antibiotic, inhibits influenza A virus infection and disease progression.

Severe respiratory disease arising from influenza virus infection has a high fatality rate. Neutrophil myeloperoxidase (MPO) has been implicated in the pathogenesis of severe influenza-induced pneumonia because extracellularly released MPO mediates the production of hypochlorous acid, a potent tissue injury factor. To search for candidate anti-influenza compounds, we screened leucomycin A3 (LM-A3), spiramycin (SPM), an erythromycin derivative (EM900, in which anti-bacterial activity has been eliminated), and clarithromycin (CAM), by analyzing their ability to inhibit MPO release in neutrophils from mice and humans. When each candidate was injected into mice infected with a lethal dose of A/H1N1 influenza virus (PR-8), LM-A3 produced the highest survival rate (80.9%). We found that LM-A3 induced beneficial effects on lung pathology and viral proliferation involved in the regulatory activity of MPO release, pro-inflammatory cytokines and interferon-α production in the lung. SPM and EM900 also induced positive survival effects in the infected mice, whereas CAM did not. We further found that these compounds inhibit virus proliferation in human pneumonia epithelial A549 cells in vitro. LM-A3 showed effective action against influenza A virus infection with high anti-viral activity in human host cells, indicating the possibility that LM-A3 is a prospective lead compound for the development of a drug for human influenza. The positive survival effect induced by EM900 suggests that pharmacological architectures between anti-bacterial and anti-influenza virus activities can be dissociated in macrolide derivatives. These observations provide valuable evidence for the potential development of novel macrolide derivatives that have strong anti-viral but no anti-bacterial activity.

Expression of Th2-skewed pathology mediators in monocyte-derived type 2 of dendritic cells (DC2).

The information conveyed from dendritic cells (DCs) to naïve CD4(+) T cells has crucial influence on their differentiation toward effector T cells. In an effort to identify DC-derived molecules directly contributing to T cell differentiation, we searched for molecules distinctively expressed between two DC subtypes, which were differentiated from peripheral monocytes by cultivation with GM-CSF (for DC1) or IL-3 (for DC2) in the presence of IL-4 and had the ability to induce naïve T cells to differentiate into Th1 or Th2 cells, respectively. As the first step to address this issue, we subtracted DC1 transcripts from those of DC2 and compiled the gene profile dominantly expressed in DC2, whose products are known to reside in other than the nucleus. Intriguingly, many of them were molecules involved in Th2-skewed disease pathologies, such as FN1, ITGAE, GPNMB, PLAUR, FPRL2, LILRB4, SERPINE1, ALOX15, TBXAS1, NCF2, CCL3, IL1RN, SPARC, and STAB1, suggesting that DCs function not only as antigen presenting cells but also as producers of Th2 pathology specific milieus leading to disease deteriorations. We also found that expressions of CYP27A1, PPAP2B, RSAD2, and ABCC3 were up-regulated in DC2, implying their significant function in Th2-deviated states. The identification of differentially expressed genes between DC subtypes provides new insights into their functions and our comparative gene expression profile will be highly useful for the identification of DC-derived key molecules for T cell differentiation.