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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.

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.

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.

Macrophages (Mphis) have various functions and play a critical role in host defense and the maintenance of homeostasis. Mphis exist in every tissue in the body, but Mphis from different tissues exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression and function, and are called by different names. However, the precise mechanism of the generation of macrophage heterogeneity is not known. In the present study, the authors examined the functional heterogeneity of Mphis generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF).CD14 positive human monocytes (Mos) were incubated with M-CSF and GM-CSF for 6-7 days to stimulate the generation of M-CSF-induced monocyte-derived Mphis (M-Mphis) and GM-CSF-induced monocyte-derived Mphis (GM-Mphis), respectively. The expression of cell surface antigens and several functions such as antigen presenting cell activity, susceptibility to oxidant stress, and the susceptibility to HIV-1 and mycobacterium tuberculosis infection were examined.GM-Mphis and M-Mphis are distinct in their morphology, cell surface antigen expression, and functions examined. The phenotype of GM-Mphis closely resembles that of human Alveolar-Mphis (A-Mphis), indicating that CSF-induced human monocyte-derived Mphis are useful to clarify the molecular mechanism of heterogeneity of human Mphis, and GM-Mphis will become a model of human A-Mphis.

Monocytes from patients with chronic myelomonocytic leukemia (CMML-Mo) strongly expressed CD14 and CD2, but not CD16. Macrophages (MΦ) generated from CMML-Mo expressed CD32, but not CD16. The expression levels of CD64 were significantly reduced from normal MΦ. While CMML-Mo-derived MΦ phagocytosed sensitized SRBC, these cells exhibited a similar defect as CMML-Mo in the ability to produce O2-. CMML-Mo could generate dendritic cells (DC), but not multinucleated giant cells (MGC), suggesting that CMML-Mo are different from normal Mo in cell surface marker expression, O2- production, and the ability to differentiate into MΦ and MGC, but not DC.

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.

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.

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.

In vivo and in vitro experiments show that polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) inhibit mitogen- or antigen-stimulated proliferation of T cells in rodents and humans. However, the exact manner and mechanisms by which PUFA inhibits T cell proliferation is not clear. In the present study, we investigated the suppressive effects of EPA, an n-3 PUFA, on PHA stimulated human peripheral blood T cells. Our results showed that EPA suppresses mitogen- or antigen-stimulated human T cell proliferation by at least 2 steps; step 1) EPA suppresses T cell proliferation by inhibiting IL-2R alpha expression and IL-2 production; step 2) EPA induces cell death of blast T cells without reducing the expression of IL-2R alpha. We also showed that EPA selectively stimulates the cell death of blast T cells but not resting T cells. The suppressive effect of EPA was mediated via the production of reactive oxygen products, because EPA-stimulated H2O2 production and the suppressive effect of EPA was restored by addition of catalase or NAC. These results taken together suggest that such immunosuppressive effects of EPA may explain the apparent benefits of EPA-enriched diets for patients with inflammatory disorders.

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.

Nalpha-[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be D by using HPLC with chiral column. For this analysis, Nalpha-(D or L)-[3-(hexadecanoyloxy)hexadecanoyl]-L-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.

ヒト単球をGM-CSFまたはM-CSF存在下に培養すると,形態,表面マーカー,機能(貪食能,活性酸素産生能,抗原呈示能,HIV感染感受性など)の異なる2種類のマクロファージ(Mφ)に分化すること,GM-CSFで分化誘導したMφは,ヒトの肺胞Mφに似ていることが知られた.またCSFによるヒト単球のMφへの分化はIL-4により修飾され,GM-CSFとIL-4によりCD1陽性の樹状細胞(DC)に,またM-CSFとIL-4によりTRAP陽性の破骨細胞様多核巨細胞(MGC)への分化が誘導されることが知られた.単球由来DCは,既にGM-CSFによりMφへ変換する能力は有していないが,M-CSFのレセプターであるc-fmsを有しM-CSFに反応してMφに分化可能である.しかしTNF-αで処理することによりc-fmsの発現が抑制されM-CSFによるMφへの分化能を失うことが知られた.また単球由来MGCの形成には内在性のIL-1とIL-6が重要な役割を果たしており,CD4/HLA-DR,LFA-1/ICAM-1及びCD14とそのリガンドの相互作用が必要なことが示唆された.

Interleukin-10 (IL-10) has been reported to be a negative cytokine for monocytes/macrophages. In the present study, we showed that IL-10 is rather a positive cytokine and augments the growth and differentiation of human monocytes stimulated with macrophage colony-stimulating factor (M-CSF ). Highly purified adherent human monocytes were cultured for 7 days with M-CSF in the presence or absence of IL-10. The number of recovered cells increased in the culture of monocytes with M-CSF + IL-10 compared to the culture with M-CSF alone. IL-10 alone was not enough to maintain the survival and differentiation of monocytes into macrophages. Morphological change cultured in M-CSF was also accelerated by addition of IL-10, and macrophages cultured in M-CSF + IL-10 were more elongated compared to macrophages cultured with M-CSF alone. Binding of 125I-M-CSF to monocytes incubated with M-CSF + IL-10 was about 1.7-fold higher than that to monocytes incubated with M-CSF alone. In accordance with the binding study, Northern blot analysis showed that the levels of the expression of c-fms, M-CSF receptor, mRNA in macrophages cultured in M-CSF + IL-10 were higher than that in macrophages cultured in M-CSF alone. Macrophages cultured in M-CSF + IL-10 expressed higher level of FcγRI, II, III, and showed augmented Fcγ receptor mediated phagocytosis. The former also produced higher level of H2O2 and O−2 , when stimulated with zymosan, and of IL-6 when stimulated with lipopolysaccharide compared to the latter. These results taken together suggest that IL-10 augments the growth and differentiation of human monocytes cultured in M-CSF.

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.

The hypothermic response of mice to ornithine-containing lipids (Orn-Ls) of the form alpha-N-(3-acyloxyacyl)-ornithine and to endotoxin (Escherichia coli 0111:B4 lipopolysaccharide [LPS]) was studied. After the administration of Orn-L or LPS to C3H/HeSlc mice, body temperature decreases were determined at 30-min intervals by inserting a thermistor into the rectum of each mouse. When Orn-L (750 microg) or LPS (70 microg) was injected into the mice, body temperature decreases of 0.8 and 2.0 degrees C, respectively, occurred 1.8 to 2.0 h later. These body temperature decreases were completely suppressed by the preadministration of indomethacin. When anti-tumor necrosis factor alpha (TNF-alpha) antibody was administered before the administration of Orn-L or LPS, only the body temperature decrease by LPS was suppressed. The body temperature decrease by Orn-L was suppressed by anti-interleukin-1beta (IL-1beta) antibody preadministration. Next, in order to study IL-1beta and TNF-alpha mRNA expression in macrophages, peritoneal macrophages were collected 40 min after the administration of Orn-L or LPS to mice. The expression of IL-1beta mRNA by stimulation with Orn-L was as strong as that by stimulation with LPS, but the expression of TNF-alpha mRNA by stimulation with Orn-L was very weak. Our previous studies of in vitro macrophage activation by Orn-L proved that strong induction of IL-1 and prostaglandin E2 generation by Orn-L occurred (Y. Kawai and K. Akagawa, Infect. Immun. 57:2086-2091, 1989). From these experiments, the weak body temperature decrease in mice caused by Orn-L was found to be mediated by cytokines different from those which mediate the strong body temperature decrease caused by LPS. Namely, it was caused by prostaglandin E2 being mediated by IL-1 but not by TNF-alpha.

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.

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 IC50value 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.

We have previously isolated a lipopolysaccharide (LPS)-resistant mutant (named LR-9) of a cultured macrophage-like cell line, J774.1. This mutant had defective LPS binding [Hara-Kuge, S., Amano, F., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 6606-6610]. In this study, we found that: (1) LPS-binding to parental J774.1 cells was dependent on a serum factor with a molecular weight of about 60 kDa, probably LPS binding protein (LBP); (2) LPS-binding to J774.1 cells was markedly reduced by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC); (3) mutant LR-9 cells were defective in LPS-binding even in the presence of serum; (4) LR-9 cells lacked CD14 protein on flow cytometric and immunoblot analyses, but retained normal CD14 mRNA levels on RNA blot analysis; (5) small amounts of LPS (1 to 10 ng/ml) activated J774.1, but not LR-9 cells, to secrete tumor necrosis factor-alpha and to release arachidonate metabolites, whereas both J774.1 and LR-9 were activated by large concentrations of LPS (100 to 1,000 ng/ml). These results provide genetic evidence that CD14 molecules in J774.1 cells play a crucial role in LPS-binding and in LPS-triggered signal transduction, and indicate that large amounts of LPS can activate J774.1 cells without the participation of CD14 molecules.

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.

The effect of various bacterial cell wall components on in vitro biological function of murine peritoneal exudate macrophages was evaluated. We examined four different parameters of metabolic activity and monokine secretion. Peritoneal exudate macrophages from rats and guinea pigs, all of the strains tested, were stimulated by whole bacterial cell wall preparations, purified bacterial cell wall peptidoglucans, its water-soluble peptidolglycan fragments, muramyl dipeptides and amphipathic substances. Murine peritoneal exudate macrophages were activated by amphipathic substances of gram-positive bacteria. However, macrophages from mice, irrespective of strains, were not stimulated in the in vitro assay systems by purified bacterial cell wall peptidoglycan, water-soluble bacterial peptidoglycan fragments or muramyl dipeptides. These results suggest that macrophage activation by bacterial peptidoglycan in vitro is animal species specific.

Summary The effect of human lung conditioned medium (HLCM) on the DNA synthesis in cultured human alveolar macrophages (HAM) was evaluated. The medium from human lung cultured for 3 days (3d-HLCM) promoted the DNA synthesis as well as the recombinant human GM-CSF does. On the other hand, that cultured for six days (6d-HLCM) did not promote the DNA synthesis but strongly suppressed GM-CSF induced DNA synthesis in HAM. This growth inhibitory effect was also observed when several macrophage like cell lines were cultured with 6d-HLCM. Partial purification of an inhibitory factor on gel permeation HPLC revealed two distinct peaks of activity with molecular weights of 38 kd and 110 kd.

Macrophages (MΦs) are a major source of HIV-1 especially in patients with tuberculosis. There are MΦs that are permissive and those that restrict HIV-1. Regulation of hematopoietic cell kinase (Hck) activity and selective expression of CCAAT enhancer binding protein β (C/EBPβ) isoforms greatly contribute to determine distinct susceptibility of MΦs to HIV-1. Resistance is attributable to reduced expression of Hck and augmented expression of an inhibitory small isoform of C/EBPβ. Derivatives of erythromycin A (EMA) EM201 and EM703 inhibit the replication of HIV-1 in tissue MΦs, at posttranscriptional and translational levels. We demonstrate that EM201 and EM703 convert tissue MΦs from HIV-1 susceptible to HIV-1 resistant through down-regulation of Hck and induction of small isoforms of C/EBPβ. These drugs inhibit p38MAPK activation which is expressed only in susceptible tissue MΦs. Activated CD4 + T cells stimulate the viral replication in HIV-1 resistant MΦs through down-regulation of small isoforms of C/EBPβ 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 MΦs 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.

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.

M-colony-stimulating factor (M-CSF)-induced monocyte-derived macrophages (M-Mphi) required continuous presence of M-CSF for their survival, and depletion of M-CSF from the culture induced apoptosis, whereas human alveolar macrophages (A-Mphi) and granulocyte-macrophage (GM)-CSF-induced monocyte-derived macrophages (GM-Mphi) survived even in the absence of CSF. The expression of BCL-2 was higher in M-Mphi, and M-CSF withdrawal down-regulated the expression. The expression of BCL-X(L) was higher in A-Mphi and GM-Mphi, and the expression was CSF-independent. The expression of MCL-1 and BAX were not different between M-Mphi and GM-Mphi and were CSF-independent. Down-regulation of the expression of BCL-2 and BCL-X(L) by RNA interference showed the important role of BCL-2 and BCL-X(L) in the survival of M-Mphi and GM-Mphi, respectively. Human erythrocyte catalase (HEC) and conditioned medium obtained from GM-Mphi or A-Mphi cultured in the absence of GM-CSF prevented the M-Mphi from apoptosis and restored the expression of BCL-2. The activity of the conditioned medium was abrogated by pretreatment with anti-HEC antibody. Anti-HEC antibody also induced the apoptosis of M-Mphi cultured in the presence of M-CSF and GM-Mphi and A-Mphi cultured in the presence or absence of GM-CSF and down-regulated the expression of BCL-2 and BCL-X(L) in these Mphis. GM-Mphi and A-Mphi, but not M-Mphi, can produce both extracellular catalase and cell-associated catalase in a CSF-independent manner. Intracellular glutathione levels were kept equivalent in these Mphis, both in the presence or absence of CSF. These results indicate a critical role of extracellular catalase in the survival of human macrophages via regulation of the expression of BCL-2 family genes.

We have been interested in the immunomodulatory effect, to promote differentiation of the human monocytic cell line THP-1 to macrophages of EM-A. We chemically modified EM-A in order to obtain derivatives with stronger promoting activity of monocyte to macrophage differentiation and no antibacterial activity. Most of the EM701 derivatives produced, all 12-membred macrolides, were remarkably active and were free of antibacterial activity. Among them, the most potent derivative was EM703, which showed very weak gastrointestinal motor-stimulating activity. EM703 may be useful tool to study the mechanisms of action of macrophage differentiation, and may be lead candidate for the development of new therapeutic drugs for chronic airway disease.

We previously demonstrated that IL-10 alone does not stimulate growth and differentiation of human monocytes, but enhances those of monocytes stimulated with M-CSF. We studied here the effect of IL-10 on human monocytes stimulated with GM-CSF. Monocytes stimulated with GM-CSF alone survived and developed into macrophages. Monocytes cultured with GM-CSF plus IL-10, however, died through apoptosis. IL-10 decreased expression of bcl-2, bcl-xL, and mcl-1- but not bax mRNA in monocytes stimulated with GM-CSF. IL-10 did not change the expression of mRNA of both GM-CSFR α-chain and β-chain, but inhibited tyrosine phosphorylation of STAT5 and extracellular signal-regulated kinases 1 and 2 in the monocytes. The inhibitory effect of IL-10 was restricted to treatment 48 h after stimulation with GM-CSF. Addition of IL-10 after that time induced neither apoptosis nor a decrease in expression of bcl-2, bcl-xL, and mcl-1 mRNA. IL-10, however, inhibited LPS-induced TNF-α production even in these cells, indicating that the cells still possessed responsiveness to IL-10. Monocytes pretreated for >48 h with GM-CSF became resistant to GM-CSF withdrawal, and the cells could survive without GM-CSF. These results indicate that IL-10 selectively inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling events induced by GM-CSF, but the timing of addition of IL-10 is critical, and IL-10 had to be added within 48 h after stimulation with GM-CSF to achieve the inhibitory effect. These results taken together with our previous results indicate that IL-10 plays a pivotal role in monocyte survival and development into macrophages in concert with M-CSF and GM-CSF.

Human alveolar macrophages (A-MPhi) and macrophages (MPhi) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MPhi) express high levels of catalase activity and are highly resistant to H(2)O(2). In contrast, MPhi generated from monocytes by macrophage colony-stimulating factors (M-MPhi) express low catalase activity and are about 50-fold more sensitive to H(2)O(2) than GM-MPhi or A-MPhi. Both A-MPhi and GM-MPhi but not M-MPhi can induce catalase expression in both protein and mRNA levels when stimulated with H(2)O(2) or zymosan. M-MPhi but not GM-MPhi produce a large amount of H(2)O(2) in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MPhi and A-MPhi but not M-MPhi are strong scavengers of H(2)O(2) via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MPhi is regulated by colony-stimulating factors during differentiation.

Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-κB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-κB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofac-tors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation. J. Leukoc. Biol. 67: 432–440; 2000.

Susceptibility to HIV infection was examined in macrophages differentiated from human monocytes by macrophage colony-stimulating factor (M-CSF) or granulocyte/macrophage colony-stimulating factor (GM-CSF). The replication of macrophage-tropic human immunodeficiency virus type-1 (HIV-1), which was determined by reverse transcriptase (RT) activity, was significantly suppressed in macrophages induced by GM-CSF (GM-type macrophages) but not in those induced by M-CSF (M-type macrophages). Multinucleated giant cells were formed only in M-type macrophages after HIV infection. However, the expression of CD4 molecules on the surface of both types of macrophages was similar and the proviral DNA was detectable in cell lysates of both macrophages, although the amount of proviral DNA in M-type macrophages was higher than that in GM-type macrophages. Many steps have been defined in HIV infection and replication, such as adsorption of HIV to the cell surface, internalization of the viral core into the cytoplasm, uncoating of viral RNA, reverse transcription and integration of proviral DNA into cellular DNA, transcription and translation of proviral DNA, assembly of viral components, and budding of virus particles. Our findings suggested that the suppression of HIV-1 replication in macrophages induced by GM-CSF is mainly due to a disturbance at certain steps of replication after synthesis of the proviral DNA. Thus, the suppression of HIV replication in GM-type macrophages may provide a model of the latency of HIV infection in vivo.

The susceptibility of monocyte-derived cultured dendritic cells (DCs) to human immunodeficiency virus (HIV) infection and their role in viral transmission in the immune response were studied in detail. We observed that highly purified cultured DCs were infected with the T-tropic Lai strain of HIV type 1 (HIV-1Lai) via the CD4 receptor, and this was followed by formation of the complete provirus as detected by PCR. HIV mRNAs were transcribed at only low levels, and virus production was undectable; however, the addition of the purified protein derivative antigen of tuberculin and of autologous resting T cells to HIV-1Lai-infected DCs but not to HIV-1Lai-infected macrophages led to massive HIV transmission and production. These data suggest that the interaction of infected DCs with T cells during the normal immune response could play an important role in the activation and expansion of HIV.

An in vitro culture system was developed that facilitates detailed studies of the interaction of Human Immunodeficiency Virus (HIV) with dendritic cells (DC). Cultured immature DC were generated from adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. These cells were non-adherent, non-phagocytic and had a veiled surface appearance. They expressed high levels of MHC class I and II proteins, CD1a, B7/BB1 and low levels of CD4, and were known to possess a potent soluble antigen presenting capacity. Upon infection with the HIV-1 strains Lai (lymphocytotropic) and BaL (monocytotropic), the viral RNA was reverse transcribed to complete DNA provirus. However the infection was non-productive as judged from measuring the activity of the virus encoded reverse transcriptase in the culture supernatant. Thus HIV infection was restricted at a step post entry.

Recent reports have suggested that long-term administration of erythromycin (EM) appears to ameliorate some of chronic inflammatory processes where macrophages and lymphocytes play important roles. Our study was initiated to examine the effect of EM on monocyte-macrophage lineage in vitro. EM (1 approximately 100 micrograms/ml) significantly increased the number of adherent monocyte-derived macrophages after 7 days of culture. The combination of EM and macrophage colony stimulating factor (M-CSF) synergistically increased the number of monocyte-derived macrophages, while the combination of EM and granulocyte-macrophage colony stimulating factor exerted an additive effect. Culture with EM induced the expression of a surface antigen CD71, one of the activation markers of macrophages as compared with control cultures. The combination of EM plus M-CSF significantly enhanced H2O2-producing capacity of those cells as compared with M-CSF alone. A differentiation process of monocytoid THP-1 cells was also augmented by EM. These results indicate that EM promotes differentiation of human monocyte-macrophage lineage, altering their functions.

Erythromycin (EM), a macrolide antibiotic has been recently reported to depress the extent of inflammation irrespective of its antimicrobial action. Our study was initiated to examine the effect of EM on T cell proliferation in vitro, since other macrolide antibiotics FK506 and rapamycin (RAP) have been well known to possess strong immunosuppressive or anti-inflammatory potential. EM had a suppressive effect on the proliferative response of human lymphocytes stimulated with mitogens and antigens, while EM had no effect on concanavalin A (Con A)-induced interleukin-2 (IL-2) production or IL-2R alpha (CD25) expression. Delayed addition of EM after the first 48 hours of mitogenic stimulation did suppress IL-2-dependent proliferation of Con A blasts, whereas pretreatment with EM for the first 48 hours of stimulation did not impede the subsequent IL-2-dependent proliferation of obtained blast cells. The results indicate that EM suppresses T cell proliferation at a late stage in the activation process by impairing their response to IL-2. This antilymphocytic action of EM was quite distinct from that of FK506 or cyclosporin A (CsA) but was similar to that of RAP. Unlike RAP, however, EM did not antagonize FK506-induced suppression but potentiated the action of FK506 and CsA. The addition of an enteric hormone motilin, a receptor of which was previously found to be occupied by EM, unaffected the lymphocyte proliferation and the subsequent EM-induced suppression. These data suggest that EM operates through an undefined mechanism probably distinct from that of FK506, CsA, RAP or motilin.

Lipoamino acids are the substances which are present as the constituents of the cell membranes of relatively wide range of bacteria. As shown in Fig 1, the structures possess both hydrophobic lipid moiety and hydrophilic amino acid moiety (Ser or Orn) (3, 4, 5). We have investigated the structures and hemagglutinating activity of some lipoamino acids from Bordetella (3), Flavobacterium (4) and Pseudomonas (5). From these studies, it was resolved that the lipoamino acids possessing the fatty acids of carbon number 16–16 or 17–15 exhibit the higher hemagglutinating activity. Serine-containing lipid was found only in Flavobacterium and was named ‘Flavolipin’ by us (4). Based on these studies, we used this time the two kinds of lipoamino acids of Flavobacterium shown in Fig 1.

In the previous study (Ikeda-Fujita et al., 1987b, 1987c) we demonstrated that a lipopolysaccharide fraction prepared from ’delipidated’ cells of Mycobacterium bovis BCG by extraction with 95% phenol/water (Westphal method) and by fractionation of the water-phase extract with Sepharose 6B and 4B columns exhibits a variety of endotoxin-like bioactivities. The fraction contained no detectable amounts of chemical markers of hitherto described immunomodulators of bacterial origin such as endotoxic lipopolysaccharides (lipid A), cord factor (trehalose mycolates), peptidoglycans (muramylpeptides), lipoteichoic acids, and others (Kotani et al., 1986).

Lipopolysaccharide (LPS) from gram-negative microorganisms is a potent activator of macrophages. LPS induces macrophages to secrete immunoregulatory substances including interleukin 1 (IL 1), tumor necrosis factor (TNF), interferon, colony stimulating factors (CSFs) and, prostaglandins. Furthermore LPS activates tumor cytotoxicity of macrophages by itself or in combination with interferon.

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate two novel glycolipids, designated X and Y. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3, and lipid Y has the same structure as X, except for the additional presence of a palmitoyl moiety on the N-linked beta-hydroxymyristate. We have examined the activities of X, Y, and several related compounds as activators of macrophages. Both X and Y induce morphological changes (spreading), prostaglandin E2 synthesis, and killing of tumor cells by mouse peritoneal macrophages in vitro, properties with which lipopolysaccharide and lipid A are also endowed. Both glycolipids have similar effects on the macrophage-like mouse cell line J774.1. Selective removal from lipid X of either the ester-linked beta-hydroxymyristate at position 3 or the phosphate at position 1 abolishes activity. Our results show that the monosaccharides X and Y retain some of the properties of intact lipopolysaccharide and lipid A with respect to macrophage activation. Because the structures of X and Y are defined, our findings should facilitate the elucidation of the molecular mechanism of macrophage activation by lipid A.

Although peritoneal resident macrophages (PRM) or peritoneal exudate macrophages (PEM) were activated by lipopolysaccharide (LPS) to kill tumor cells in vitro, lung macrophages (LM) obtained by mincing lung tissues or by harvesting bronchial lavage were not activated by LPS under any experimental conditions, i.e., different LPS concentrations, incubation times and cytotoxicity assay methods. The unresponsiveness of LM to LPS was seen in all of the mouse strains tested. Treatment of LM with indomethacin did not affect the unresponsiveness, although it greatly augmented the cytotoxicity of PRM stimulated with LPS. LM treated in vitro with crude lymphokines (LK) did not show cytotoxicity, but became sensitive to LPS and cytotoxic for tumor cells. LM treated first with crude LK and then with LPS were cytotoxic, but LM treated first with LPS and then with crude LK were not. The ability of crude LK to render LM responsive to LPS was neutralized by rabbit anti-mouse gamma interferon (IFN-gamma) antiserum but not by anti-mouse IFN-(alpha + beta) antiserum. LM treated with recombinant murine IFN-gamma became responsive to LPS and showed cytotoxicity. LM were resistant to direct toxicity of LPS under conditions in which significant populations of PRM and PEM died. However, LM became sensitive to direct toxicity of LPS by treatment with crude LK or recombinant murine IFN-gamma. Fluorescence microscopy showed that almost all PRM and PEM were stained with fluorescein isothiocyanate (FITC)-LPS, while less than 5% of the LM were stained. Instead, approximately 60% of the LM treated with the crude LK or recombinant IFN-gamma for 20 h were stained with FITC-LPS. Fluorescence-activated cell sorter (FACS) analysis confirmed this result. The staining of IFN-gamma treated LM with FITC-LPS was inhibited by polymyxin B or unlabeled LPS. These results suggest that the defective responsiveness of LM to LPS is due to the lack or very low expression of LPS-binding sites on the cell surface and that in vitro treatment with IFN-gamma brings about the expression of them and renders LM responsive to LPS.

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.

Monocyte-derived cultured dendritic cells (DCs) are potent antigen-presenting cells (APCs) and are susceptible to HIV-1Laiinfection. Compared to the low level of virus production by HIV-1-infected DCs alone, a level of virus two to three orders of magnitude higher was produced by cocultivation of HIV-1-infected DCs with autologous resting CD4+T cells in the presence of a nominal antigen. In this coculture system, direct contact of HIV-1-infected DCs with T cells was crucial for efficient virus transmission and subsequent virus production. Blocking of the LFA-1/ICAM-1 or LFA-3/CD2 interaction between these cells substantially reduced virus production, without influence on IL-2 production by activated T cells. In contrast, cell–cell transmission of HIV between non-APCs and activated T cells was not blocked by an antibody against LFA-3. Since a low level of virus production by HIV-infected DCs was upregulated by cross-linking of CD40, it was suggested that not only focal adhesion, but also mutual activation of HIV-infected DCs and T cells through adhesion molecules, may potentiate virus transmission and production and that such activation signals to HIV may be distinct from signals responsible for IL-2 production in activated T cells.

The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing β-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O 2 − generation in mouse peritoneal macro-phages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3- O -β-hydroxymyristoyl group from lipid X completely abolished these effects, suggesting that both 1-phosphate and 3- O -β-hydroxymyristoyl groups are essential for the elevated O 2 − generation and induction of turmoricidal activity due to lipid X.