Human alveolar macrophages suppress interleukin-1 (IL-1) activity via the secretion of prostaglandin E2

It has been suggested that human alveolar macrophages have a limited capacity to release interleukin-1 (IL-1). To determine whether this apparent defect in cell function is related to the release of factors that inhibit the response of lymphocytes to IL-1, we evaluated the capacity of human alveolar macrophages to release prostaglandin E2 (PGE2), a factor that is known to suppress the response of lymphocytes to IL-1. The amount of PGE2 released by alveolar macrophages was dependent on the amount of LPS present in the cultures and the amount of time the cells were present in culture. After 24 h in culture, the alveolar macrophage supernatants contained sufficient amounts of PGE2 to significantly suppress PHA-induced lymphocyte proliferation (p less than 0.01), IL-1-induced thymocyte proliferation (p less than 0.001), but not IL-2-induced lymphocyte proliferation (p greater than 0.2). Consistent with these observations, only small amounts of IL-1 activity could be detected in 24-h supernatants of LPS-stimulated alveolar macrophages using thymocyte proliferation as an assay for IL-1. Using a more sensitive assay for IL-1, however, it was demonstrated that the supernatants of LPS-stimulated macrophages contained amounts of IL-1 that were not significantly different from those present in supernatants of LPS-stimulated monocytes. Indomethacin (1 microgram/ml) completely suppressed the release of PGE2 by alveolar macrophages. These observations suggest that the apparent defect in the release of IL-1 by human alveolar macrophages may be due in part to the release of large amounts of PGE2, which suppresses various lymphocyte functions.     

Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo

Objective

Macrophages show extreme heterogeneity and different subsets have been characterized by their activation route and their function. For instance, macrophage subsets are distinct by acting differently under pathophysiological conditions such as inflammation and cancer. Macrophages also contribute to angiogenesis, but the role of various specific subsets in angiogenesis has not been thoroughly investigated.

Methods and results

Matrigel supplemented with macrophage subsets [induced by IFNγ (M1), IL-4 (M2a) or IL-10 (M2c)] was injected subcutaneously in C57BL/6 J mice and analyzed by CD31 staining after 14 days. Increased numbers of endothelial cells and tubular structures were observed in M2-enriched plugs compared to control and other subsets. Additionally, more tubular structures formed in vitro in the presence of M2 macrophages or their conditioned medium. To identify a mechanism for the pro-angiogenic effect, gene expression of angiogenic growth factors was analyzed. Induced expression of basic fibroblast growth factor (Fgf2), insulin-like growth factor-1 (Igf1), chemokine (C–C motif) ligand 2 (Ccl2) and placental growth factor (Pgf) was observed in M2 macrophages. Using a blocking antibody of PlGF to inhibit M2c induced angiogenesis resulted in mildly reduced (40 %) tube formation whereas neutralization of FGF-2 (M2a) signaling by sFGFR1-IIIc affected tube formation by nearly 75 %.

Conclusions

These results indicate that macrophages polarized towards an M2 phenotype have a higher angiogenic potential compared to other subsets. Furthermore, we propose FGF signaling for M2a- and PlGF signaling for M2c-induced angiogenesis as possible working mechanisms, yet, further research should elucidate the exact mechanism for M2-induced angiogenesis.     

Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10)

We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system.     

T-helper cell type 2 (Th2) memory T cell-potentiating cytokine IL-25 has the potential to promote angiogenesis in asthma

IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25(+), IL-25R(+), and CD31(+) (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25(+), IL-25R(+), and CD31(+)/IL-25R(+) cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25(+) cells correlated inversely with the forced expiratory volume in 1 s (r = -0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25-induced angiogenesis, and the inhibitors also reduced IL-25-induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.     

MyD88 is essential to sustain mTOR activation necessary to promote T helper 17 cell proliferation by linking IL-1 and IL-23 signaling

Myeloid differentiation primary response protein 88 (MyD88) is classically known as an adaptor, linking TLR and IL-1R to downstream signaling pathways in the innate immune system. In addition to its role in innate immune cells, MyD88 has been shown to play an important role in T cells. How MyD88 regulates helper T-cell differentiation remains largely unknown, however. Here we demonstrate that MyD88 is an important regulator of IL-17-producing CD4⁺ T helper cells (Th17) cell proliferation. MyD88-deficient CD4⁺ T cells showed a defect in Th17 cell differentiation, but not in Th1 cell or Th2 cell differentiation. The impaired IL-17 production from MyD88-deficient CD4⁺ T cells is not a result of defective RAR-related orphan receptor γt (RORγt) expression. Instead, MyD88 is essential for sustaining the mammalian target of rapamycin (mTOR) activation necessary to promote Th17 cell proliferation by linking IL-1 and IL-23 signaling. MyD88-deficient CD4⁺ T cells showed impaired mTOR activation and, consequently, reduced Th17 cell proliferation. Importantly, the absence of MyD88 in T cells ameliorated disease in the experimental autoimmune encephalomyelitis model. Taken together, our results demonstrate that MyD88 has a dual function in Th17 cells by delivering IL-1 signaling during the early differentiation stage and integrating IL-23 signaling to the mTOR complex to expand committed Th17 cells.Myeloid differentiation primary response protein 88 (MyD88) was originally isolated as a cloned cDNA that was induced in M1 myeloblastic leukemia cells on activation with IL-6 (1). The function of MyD88 was then uncovered, because the C-terminal portion of MyD88 was found to be similar to the Drosophila Toll receptor and the mammalian IL-1 receptor (IL-1R) (2). This conserved region in the cytoplasmic tails of IL-1R and Toll-like receptor (TLR) is referred to as the Toll/IL-1R (TIR) domain. MyD88 is now known to play an essential role in the innate immune response by linking members of the TLR and IL-1R superfamily to the downstream activation of NF-κB and MAPKs (3).Among cytokines produced by activated innate immune cells, IL-23 has been shown to promote production of the proinflammatory cytokine IL-17 in activated T cells (4). IL-17–producing CD4⁺ T helper (Th17) cells were identified after the discovery that IL-23 is linked to traditionally Th1-associated autoimmune disorders, such as experimental autoimmune encephalitis (EAE). Il23a^−/− mice, but not Il12a^−/− mice, were shown to be autoimmune-resistant (5). IL-23 is required for Th17 cell-mediated autoimmune disorders in vivo, but the role of IL-23 in Th17 cell differentiation remains controversial (6). Previous studies support the idea that IL-23 helps expand or maintain Th17 cells (7–9). In addition, a recent study reemphasized the importance of IL-23 in the generation of pathogenic Th17 cells, showing that they can be generated with IL-23, IL-6, and IL-1β (10).In addition to IL-23, other cytokines, including TGF-β, IL-6, and IL-1, play roles in Th17 cell development. TGF-β with proinflammatory cytokines was shown to be critical in the support of Th17 cell differentiation (8, 11, 12). In particular, an inflammatory cytokine, IL-6, favors Th17 cell development by inhibiting regulatory T cells (13). The role of IL-1 in Th17 cell differentiation has been investigated as well. IL-1 receptor type 1-deficient (Il1r1^−/−) mice showed a lower incidence of EAE and severe defects in the induction of IL-17–producing T cells (14). IL-1 signaling in T cells was further shown to be involved in early Th17 cell differentiation by regulating IFN regulatory factor 4 and RAR-related orphan receptor γt (RORγt) (15).Although roles for MyD88 in the innate immune system are well established, little is known about their potential function in the adaptive immune system. Several studies have demonstrated important roles of MyD88 in T cells. For instance, T-cell expression of MyD88 is required for resistance to Toxoplasma gondii (16); the MyD88-dependent signaling pathway in CD4⁺ T cells has been shown to enhance proliferation and augment humoral immune responses (17); and MyD88 is required for T-cell effector function in the development of inflammatory bowel disease (18). Interestingly, although Myd88^−/− T cells were found to exhibit decreased IL-17 production (18), how T-cell differentiation could be regulated by MyD88 was not clear in that study.Here we investigated the molecular mechanism by which MyD88 regulates CD4⁺ T-cell differentiation. Our results demonstrate that MyD88 contributes to Th17 cell differentiation, but not to Th1 or Th2 cell differentiation. Both IL-1 and IL-23 signaling depend on MyD88 and result in up-regulation of IL-23R. MyD88-deficient Th17 cells show reduced IL-23R expression and mTOR activation, leading to impaired Th17 cell proliferation. Furthermore, MyD88 is crucial for proper Th17 cell differentiation in vivo. Thus, our findings reveal a unique role for the innate adaptor MyD88 in the regulation of Th17 cell differentiation.     

Human immunodeficiency virus type 2 (HIV2)

In the mid 1980's a second human retrovirus, capable of causing the acquired immunodeficiency syndrome (AIDS), was isolated from patients of West African origin. This virus, now called human immunodeficiency virus type 2 (HIV2), was found to be distinct from human immunodeficiency virus type 1 (HIV1) but closely related to simian immunodeficiency viruses (SIV). Although the genomes of HIV1 and HIV2 are similar there are significant differences in nucleotide and amino acid sequences, most marked with the envelope genes and proteins. Both viruses, however, bind to the same CD4 cellular receptor. HIV2 is largely confined to West Africa where it is the dominant HIV, though patients infected with HIV2 have been described in Europe and America. Its transmission, clinical features and immunological effects are similar to those associated with HIV1 infection. However, there is some suggestion that the incubation period from infection to clinical disease may be longer than with HIV1 and that HIV2 may be less pathogenic. Patients with sera that react with both HIV1 and HIV2 antigens have been described, but it is unclear whether this represents serological cross reactivity or true double virus infection. Testing for HIV2 antibodies may become increasingly necessary in HIV2 non-endemic areas.     

Associations between interleukin-1 (IL-1) gene variations or IL-1 receptor antagonist levels and the development of type 2 diabetes

Abstract. Luotola K, Pietilä A, Zeller T, Moilanen L, Kähönen M, Nieminen MS, Kesäniemi YA, Blankenberg S, Jula A, Perola M, Salomaa V (Helsinki University Hospital, Helsinki; National Institute for Health and Welfare, Helsinki, Finland; University Medical Center Mainz, Johannes Gutenberg University Mainz, Mainz, Germany; University Hospital of Kuopio, Kuopio; Tampere University Hospital and Medical School, University of Tampere, Tampere; University of Oulu and Clinical Research Center, Oulu University Hospital, Oulu; National Institute for Health and Welfare, Turku; and Institute for Molecular Medicine Finland, Helsinki, Finland). Associations between interleukin‐1 (IL‐1) gene variations or IL‐1 receptor antagonist levels and the development of type 2 diabetes. J Intern Med 2010; 269 : 322–332. Objectives. To examine whether interleukin‐1 receptor antagonist (IL‐1Ra) is a predictor for clinically incident diabetes in subjects with metabolic syndrome (MetS) and whether its predictive power is independent of C‐reactive protein (CRP), an established marker of inflammation. We further examined whether genetic variants at the interleukin‐1 (IL‐1) locus would predict clinically incident diabetes. Design. Two observational prospective cohort studies. Setting. Two separate cohorts, Health 2000 and FINRISK 1997, followed up for an average of 7.1 and 10.8 years, respectively. Subjects. Random population samples consisting of 5511 subjects aged 30–74 years in Health 2000 and 7374 subjects aged 25–74 years in FINRISK 1997. Results. During follow‐up, 141 cases of clinically incident diabetes were observed amongst subjects with MetS at baseline in Health 2000 and 248 cases in FINRISK 97. After adjustment for multiple traditional risk factors of diabetes, including age and body mass index, IL‐1Ra was a significant (P < 0.01) predictor of incident diabetes amongst men in both cohorts and amongst women in FINRISK 1997. Further adjustment for CRP reduced the hazard ratios only slightly. Genetic analyses produced nominally significant associations for three single‐nucleotide polymorphisms: rs3213448 in IL‐1 receptor antagonist (IL1RN), rs1143634 in IL‐1 beta (IL1B) and rs1800587 in IL‐1 alpha (IL1A). The two latter variants had an interaction with gender (P = 0.023 and 0.002, respectively) suggesting the presence of gender‐specific associations with the risk of clinically incident diabetes. Conclusions. IL‐1Ra predicted the progression of MetS to clinically incident diabetes independently of CRP and other risk factors. Genetic variation in the IL‐1 locus may have gender‐specific associations with the risk of type 2 diabetes.     

Interleukin (IL)-12 and IL-23 are key cytokines for immunity against Salmonella in humans

Patients with inherited deficiency of the interleukin (IL)-12/IL-23-interferon (IFN)- gamma axis show increased susceptibility to invasive disease caused by the intramacrophage pathogens salmonellae and mycobacteria. We analyzed data on 154 patients with such deficiency. Significantly more patients with IL-12/IL-23-component deficiency had a history of salmonella disease than did those with IFN- gamma -component deficiency. Salmonella disease was typically severe, extraintestinal, and caused by nontyphoidal serovars. These findings strongly suggest that IL-12/IL-23 is a key cytokine for immunity against salmonella in humans and that IL-12/IL-23 mediates this protective effect partly through IFN- gamma -independent pathways. Investigation of the IL-12/IL-23-IFN- gamma axis should be considered in patients with invasive salmonella disease.     

Antibody responses to Haemophilus influenzae type b polysaccharide vaccine in relation to Km(1) and G2m(23) immunoglobulin allotypes

The antibody responses of children immunized with Haemophilus influenzae type b polysaccharide vaccine were examined in relation to the absence or presence of the Km(1) or G2m(23) immunoglobulin allotype. Ninety-seven children, 12-83 months of age, were immunized. Sera were obtained before immunization and two months later. Total serum antibody to the type b capsule was detected by a radioactive antigen-binding assay. IgG and IgM antibody responses were measured by enzyme-linked immunosorbent assay. Antibody responses to the type b capsule were more than threefold higher in blacks with the Km(1) immunoglobulin allotype compared with those in blacks lacking this allotype (P less than .02). The isotype affected was IgG (P less than .01) and not IgM. Serum concentrations of IgG2, but not of IgG1, also were higher in blacks with Km(1) (P less than .003). In whites there were no significant differences in the total or IgG-specific antibody responses to the type b capsule in relation to the Km(1) or G2m(23) allotype.     

Neuropeptides (sp and CGRP) augment IL-1 Beta production in hsv-infected macrophages

Neuropeptides, possessing specific and functional receptors on various cells of the immune system, have the potential to regulate immune responses; and the macrophages as important components of defense against various agents, are at their influence. In this study the effect of neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) on IL-1 beta production by herpes simplex type-1 (HSV-1)-infected and also uninfected mouse peritoneal macrophages were considered. Each neuropeptide separately has upregulated IL-1 beta production by HSV- 1 infected macrophages with the greatest effect at the concentrations of 1 09M for both SP and CGRP, but no synergistic effect on IL-1 production has been observed in the presence of both neuropeptides at optimal concentrations. IL-113 production by uninfected macrophages was also moderately enhanced in the presence of each neuropeptide, but not in the presence of both neuropeptides simultaneously. It can be concluded that IL-1 beta production, which is part of macrophage mediated inflammatory response to HSV-l, is enhanced by specific doses of neuropeptides.     

Interleukin-1 (IL-1) directly and indirectly promotes hematopoietic cell growth through type I IL-1 receptor

Interleukin-1 (IL-1) has been shown to stimulate hematopoietic progenitor cell growth both in vitro and in vivo. Although IL-1 alone lacks the ability to promote hematopoietic progenitor growth in vitro, it is a potent synergistic factor in combination with other colony- stimulating factors (CSFs). Because it was unknown whether type I (p80), type II (p68), or other IL-1-binding proteins mediated the synergistic effects of IL-1 on purified progenitor cells, we used the difference in immunoreactivity between type I and type II IL-1 receptor (IL-1R) to better assess the role of these receptors in hematopoietic progenitor growth. Therefore, the synergistic effects of IL-1 alpha on IL-3-, CSF-1-, and granulocyte macrophage (GM)-CSF-induced progenitor growth, both in CFU-c and single-cell assays, were determined in the presence of monoclonal antibodies (MoAbs) 35F5 and 4E2 that block the binding of IL-1 alpha to type I and type II IL-1R, respectively. The synergistic effect of IL-1 alpha on IL-3 responsive Lin- and Lin(-)-Thy- 1+ progenitors was indirectly mediated and could be inhibited by MoAb 35F5. In contrast, IL-1 alpha directly synergized with CSF-1 and GM-CSF to promote progenitor cell growth. The direct synergistic effect of IL- 1 alpha on CSF-1-induced progenitor growth was observed in all progenitor populations examined (Lin-, Lin-Thy-1+, and Lin-Thy-1-) and was inhibited by MoAb 35F5. However, the direct synergistic effect of IL-1 alpha on GM-CSF-responsive progenitors. Lin- and Lin-Thy-1+, was partially inhibited by MoAb 35F5. In contrast, the MoAb antitype II IL- 1R (MoAb 4E2) could not inhibit the direct synergistic effects of IL-1 alpha on CSF-1- or GM-CSF-induced progenitor growth. Thus, IL-1 alpha directly and indirectly stimulates the growth and differentiation of purified progenitors through the type I IL-1R but not the type II IL-1R.     

Detection of IL-13, IL-10, and IL-6 in the leprosy skin lesions of patients during prednisolone treatment for type 1 (T1R) reactions

This study demonstrates the presence of IL-10 and IL-6, by immunohistochemistry, in the skin lesions of patients with Type 1 reactions. Fifteen patients with Type 1 reaction from Hyderabad, India were included in this study. They were all receiving standardized treatment for Type 1 reactions: a reducing course of daily oral prednisolone for 6 months. Biopsies were taken before treatment and during treatment at weeks 1, 4, and 6 months. IL-13 was observed in the lesions of most patients. By week 4 of treatment, the presence of IL-13, IL-10, and IL-6 in the lesions had decreased significantly.Although some patients showed significant clinical skin sign improvement within one week of therapy, no concomitant decrease or increase in any of the cytokines was observed at this time point. Interestingly, some cytokine activity within the lesions was observed after 6 months of treatment.     

Cellular immunity to human immunodeficiency virus type 1 (HIV-1) clades: relevance to HIV-1 vaccine trials in Uganda

The first prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine trial in Africa, with a clade B immunogen, is currently under way in Uganda, in a region where clades A and D are endemic. The use of a B clade vaccine is based on anticipated cross-recognition of endemic strains of HIV-1 in Uganda, but, in fact, little is known about the cytotoxic T lymphocyte (CTL) responses in that region. Seventeen HIV-1-infected volunteers from Kampala, Uganda, were studied to determine the immune responses elicited by natural infection with local HIV-1 strains. Despite the presence of broad cross-clade recognition, the CTL responses to the infecting viral clade were highest in most people. Recognition of nonendemic clade B antigens was similar to that of the coendemic local clade, and, in some instances, cross-recognition of clade B was greater. Nevertheless, the degree of cross-clade cellular responses we observed lends justification to the use of clade B-based immunogens in the current phase 1 vaccine trial in Uganda.     

Glycoprotein 2 (GP2): grabbing the FimH bacteria into M cells for mucosal immunity

Membranous (M) cells are specialized epithelial antigen-transporting cells scattered in the follicle-associated epithelium covering the gut lymphoid follicles such as Peyer's patches. Although the importance of M cells as a main portal for luminal antigens has long been recognized, molecular mechanisms for M-cell antigen uptake has remained largely elusive. We have recently found that glycoprotein 2 (GP2) is exclusively expressed on M cells among intestinal epithelial cells and serves as an uptake receptor for a subset of commensal and pathogenic bacteria. GP2 interacts with FimH, a major component of the type 1 pilus on the outer membrane of a subset of gram-negative enterobacilli such as E. coli and Salmonella enterica. Furthermore, GP2-FimH interaction is necessary for efficient uptake of FimH(+) bacteria by M cells and subsequent bacteria-specific mucosal immune responses. Pancreatic GP2 may also be involved in innate immunity by 'opsonization' of FimH(+) bacteria to facilitate their egestion in feces as well as translocation across the intestinal epithelium.     

Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.