Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1beta processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1beta. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1beta processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1beta processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1beta processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1beta processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1beta. Pyrin induced IL-1beta processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1beta processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1beta processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1beta processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1beta processing and release.     

Regulation of aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages.

IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.     

Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Alveolar macrophages differ from blood monocytes in human IL-1 beta release. Quantitation by enzyme-linked immunoassay

Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Influence of Phthalates on in vitro Innate and Adaptive Immune Responses

Phthalates are a group of endocrine disrupting chemicals, suspected to influence the immune system. The aim of this study was to investigate the influence of phthalates on cytokine secretion from human peripheral blood mononuclear cells. Escherichia coli lipopolysaccharide and phytohemagglutinin-P were used for stimulation of monocytes/macrophages and T cells, respectively. Cells were exposed for 20 to 22 hours to either di-ethyl, di-n-butyl or mono-n-butyl phthalate at two different concentrations. Both diesters were metabolised to their respective monoester and influenced cytokine secretion from both monocytes/macrophages and T cells in a similar pattern: the secretion of interleukin (IL)-6, IL-10 and the chemokine CXCL8 by monocytes/macrophages was enhanced, while tumour necrosis factor (TNF)-α secretion by monocytes/macrophages was impaired, as was the secretion of IL-2 and IL-4, TNF-α and interferon-γ by T cells. The investigated phthalate monoester also influenced cytokine secretion from monocytes/macrophages similar to that of the diesters. In T cells, however, the effect of the monoester was different compared to the diesters. The influence of the phthalates on the cytokine secretion did not seem to be a result of cell death. Thus, results indicate that both human innate and adaptive immunity is influenced in vitro by phthalates, and that phthalates therefore may affect cell differentiation and regenerative and inflammatory processes in vivo.     

Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2

Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-α and high number of IL-10⁺ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-α and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.     

Role of IL-15 on monocytic resistance to human herpesvirus 6 infection

Interleukin-15 (IL-15) is a cytokine that possesses a variety of biological functions, including stimulation and maintenance of cellular immune responses. Recently, it has been demonstrated that Human Herpes virus type 6 (HHV-6) enhances NK activity of human PBMC by inducing IL-15. HHV-6 is a typical immunosuppressive agent, as suggested by its tropism for both CD4+ and CD8+ T cells, B cells, monocytes/macrophages, megakaryocytes and NK cells. Moreover, several studies have indicated that mononuclear phagocyte resistance to virus infection is influenced by the cellular differentiation state. This paper describes the effect of pretreatment "in vitro" with IL-15 on the resistance of human monocytes (HM) to HHV-6 infection. Our results demonstrate that undifferentiated HM were highly resistant to HHV-6 infection, whereas HM pretreated with human recombinant IL-15 showed an increased permissiveness for HHV-6 infection. This permissiveness was characterised by higher release of extracellular virus as well as an increased percentage of antigen positive cells. Moreover, we evaluated IL-15 production after the addition of HHV-6 to monocytes precultured in different experimental conditions. Our data indicate that HHV-6-induced IL-15 production by human monocytes is not affected by the condition of "in vitro" precultivation/differentiation. Furthermore, the neutralization of IL-15 induced by HHV-6 in differentiated monocytes did not affect viral replication. These findings suggest that IL-15 acts only on the mechanisms of cellular differentiation, rendering HM more susceptible to HHV-6 infection, without interfering with virus replication.     

IL-4 inhibits macrophage-mediated killing of Plasmodium falciparum in vitro. A possible parasite-immune evasion mechanism.

Although a number of mechanisms have been put forward for immunity to malaria, their importance remains to be clarified. One of the important findings is that nonactivated monocytes and macrophages showed marked antiplasmodial activity in vitro. Recently we postulated that parasites may induce host factors that may depress the natural antiplasmodial activity of monocytes. In this investigation we identify IL-4 as a lymphokine that could function in this capacity. Human monocytes and macrophages in the absence of antiplasmodial antibody showed substantial killing of the asexual erythrocytic forms of Plasmodium falciparum as determined by a radiometric assay. Suppression of this killing was seen if the mononuclear phagocytes were pretreated with human rIL-4 at concentrations of 10 to 250 U with optimum activity between 100 and 250 U/2 x 10(5) cells. Cells from some individuals were rendered completely inactive by the IL-4 treatment. In contrast, IL-4 did not affect the neutrophil-mediated anti-P. falciparum activity. Our work identifies a potentially important parasite immune evasion mechanism involving IL-4 suppression of macrophage antiparasite activity.     

The stimulation of the bactericidal activity of human neutrophils by lymphoid cells activated by interleukin-2; the effect of inflammatory cytokines]

Regulation of bactericidal activity of neutrophils (BAN) of healthy volunteer blood donors was studied. Interleukin-2 (IL-2)-activated lymphocytes potentiated BAN more effectively then resting lymphocytes. IL-2-activated mononuclear cells (containing lymphocytes and monocytes/macrophages) decreased neutrophil-potentiating activity when compared with nonactivated mononuclear cells. It was concluded that IL-2-activated monocytes exerted potent suppressive influence upon lymphocytes. Recombinant interleukin-1 beta, tumour necrosis factor-alpha, interferon-gamma acted synergistically with IL-2-activated lymphocytes on BAN when the level of neutrophil bactericidal activity was low.     

Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.     

Increased Escherichia coli-Induced Interleukin-23 Production by CD16+ Monocytes Correlates with Systemic Immune Activation in Untreated HIV-1-Infected Individuals

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16⁺ monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14⁺ monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16⁺ monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16⁺ monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.     

Interleukin-12 p40 mRNA Expression in Bovine Leukemia Virus-Infected Animals: Increase in Alymphocytosis but Decrease in Persistent Lymphocytosis

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

The uptake of apoptotic cells drives Coxiella burnetii replication and macrophage polarization: a model for Q fever endocarditis

Patients with valvulopathy have the highest risk to develop infective endocarditis (IE), although the relationship between valvulopathy and IE is not clearly understood. Q fever endocarditis, an IE due to Coxiella burnetii, is accompanied by immune impairment. Patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes, as determined by the measurement of active caspases and nucleosome determination. The binding of apoptotic cells to monocytes and macrophages, the hosts of C. burnetii, may be responsible for the immune impairment observed in Q fever endocarditis. Apoptotic lymphocytes (AL) increased C. burnetii replication in monocytes and monocyte-derived macrophages in a cell-contact dependent manner, as determined by quantitative PCR and immunofluorescence. AL binding induced a M2 program in monocytes and macrophages stimulated with C. burnetii as determined by a cDNA chip containing 440 arrayed sequences and functional tests, but this program was in part different in monocytes and macrophages. While monocytes that had bound AL released high levels of IL-10 and IL-6, low levels of TNF and increased CD14 expression, macrophages that had bound AL released high levels of TGF-beta1 and expressed mannose receptor. The neutralization of IL-10 and TGF-beta1 prevented the replication of C. burnetii due to the binding of AL, suggesting that they were critically involved in bacterial replication. In contrast, the binding of necrotic cells to monocytes and macrophages led to C. burnetii killing and typical M1 polarization. Finally, interferon-gamma corrected the immune deactivation induced by apoptotic cells: it prevented the replication of C. burnetii and re-directed monocytes and macrophages toward a M1 program, which was deleterious for C. burnetii. We suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for bacterial persistence.     

Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.     

Cytomegalovirus infection of peripheral blood mononuclear cells: effects on interleukin-1 and -2 production and responsiveness.

Cytomegalovirus suppresses the proliferative response of peripheral blood mononuclear cells to phytohemagglutinin. In these experiments, we identified which mononuclear cell subpopulation might be responsible for the suppression. We found that prior infection of either lymphocytes or monocytes followed by reconstitution with monocytes or lymphocytes, respectively, would abrogate the proliferative response in a subsequent culture with phytohemagglutinin. Infection of either cell type also reduced both the production of interleukin-1 (IL-1) and IL-2 and the proliferative response to exogenously supplied IL-1 or IL-2. We did not find evidence for an IL-2 antagonist. These experiments suggest that cytomegalovirus causes a metabolic derangement in lymphocytes and monocytes and impairs their ability both to produce and to respond to physiological mediators of the immune response.