Selective induction of monocyte and not neutrophil-attracting chemokines after influenza A virus infection.

It is characteristic for virus infections that monocytes/macrophages and lymphocytes infiltrate infected tissue while neutrophils are absent. To understand the mechanisms selectively attracting mononuclear cells in viral diseases, we examined in an influenza A virus model the expression and regulation of chemokines as candidate molecules responsible for the immigration of leukocytes into inflamed tissue. After influenza A virus infection of human monocytes, a rapid expression of the mononuclear cell attracting CC-chemokine genes MIP-1, MCP-1, and RANTES occurred which was followed by the release of chemokine proteins. In striking contrast to CC-chemokines, the expression of the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha was completely suppressed after influenza A infection. The release of other neutrophil chemotactic factors was excluded by microchemotaxis assays. These results suggest that the virus-specific induction of mononuclear cell-attracting chemokines accounts for the preferential influx of mononuclear leukocytes into virus-infected tissue.

Stimulation of heme oxygenase in macrophages and liver by endotoxin.

In rat peritoneal macrophages, engaged in erythrophagocytosis in vitro, endotoxin stimulated heme oxygenase (HO) activity, which was additive to the substrate-mediated enzyme induction produced by the ingested erythrocyte hemoglobin. Endotoxin neither appeared to injure the erythrocytes, nor did it enhance the rate of erythrophagocytosis. In intact rats, HO activity in both parenchymal and sinusoidal cells of the liver was increased after treatment with endotoxin. It is likely that endotoxin directly stimulates HO activity, a process which may account for the reported rise in bilirubin formation in endotoxin-treated animals. The effect of endotoxin on HO may represent part of the general activation of phagocytic cells by endotoxin.

Increase of CCR1 and CCR5 expression and enhanced functional response to MIP-1 alpha during differentiation of human monocytes to macrophages.

Chemokines and their receptors regulate migration of leukocytes under normal and inflammatory conditions. In this study, we analyzed the CC chemokine receptor (CCR) expression of monocytes differentiating in vitro to macrophages. We observed a time-dependent change of expression and functional responsiveness of CCR1, CCR2, and CCR5 within 48 h. Whereas freshly harvested monocytes were strongly attracted by monocyte chemotactic protein 1 (MCP-1), a specific ligand for CCR2, only a weak response was observed to macrophage inflammatory protein 1alpha (MIP-1alpha), which binds to CCR1 and CCR5. In striking contrast, differentiated macrophages displayed a strong chemotactic response to MIP-1alpha and only a weak response to MCP-1. These findings were paralleled by intracellular calcium shifts. During the time course of monocyte to macrophage differentiation, mRNA levels and surface expression of CCR2 decreased, whereas that of CCR1 and CCR5 increased. The time-dependent switch from CCR2 on monocytes to CCR1 and CCR5 on mature macrophages reflects a functional change belonging to the differentiation process of monocytes to macrophages and may form the basis for a differential responsiveness of monocytes and macrophages to distinct sets of chemokines.

Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response.

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4-6 h. Most macrophages died by apoptosis within 25-30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, interferon (IFN)-alpha/beta, and CC-chemokines. The basic mechanisms of virus-induced cytokine expression are still unknown and appear to involve transcription factors such as nuclear factor-kappaB and AP-1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES were produced while the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha were entirely suppressed. This selective induction of CC-chemokines may explain the preferential influx of mononuclear leukocytes into virus-infected tissue. Our data show that monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen-specific defense.

Activation of macrophages by lymphokines from T-cell clones: evidence for different macrophage-activating factors.

The data reported in this paper demonstrate that macrophage-activating factors (MAFs) are a heterogeneous group of T-cell-derived lymphokines. Two long-term T-cell clones, Cl 96 and PK 7.1.2E8, were potent sources of MAFs (MAF96 and MAF7.1.2E8). These MAFs could be distinguished by differential activation of macrophages. Activation of resident murine macrophages with MAF7.1.2E8 enhanced RNA and glycoprotein synthesis, hexosemonophosphate shunt (HMPS) activity, release of oxygen metabolites (O-2 and H2O2), pinocytosis and tumor cytostasis, whereas no effect on schistosomula killing and tumor cytolysis could be observed. In contrast, MAF96 enhanced glycoprotein synthesis, HMPS activity, release of oxygen metabolites and prostaglandin E, schistosomula killing, and tumor cytostasis and cytolysis, while RNA synthesis and pinocytosis were decreased. These findings show that MAFs from both T-cell clones share some properties but markedly differ in others. In addition, the macrophage-activating properties of MAF96 but not of MAF7.1.2E8 could selectively be inhibited by a rabbit anti-lymphokine antiserum. This demonstrates a serological difference between MAF activities from both clones. Although at optimal concns both MAFs were active in the absence of lipopolysaccharide (LPS), the activity of suboptimal doses of MAF96 but not of MAF7.1.2E8 could be enhanced by LPS. These findings show that different MAFs from T-cell clones may be useful to clarify molecular mechanisms of macrophage activation.

Serum amyloid A (SAA) protein enhances formation of cyclooxygenase metabolites of activated human monocytes.

As serum amyloid A (SAA), an apolipoprotein associated with HDL during the acute-phase reaction may induce Ca2+ mobilization in human monocytes we raised the question whether SAA1 the predominant isoform of human acute-phase SAA is able to alter eicosanoid formation. In resting monocytes SAA1 was without effect on the secretion of cyclooxygenase metabolites while in calcium ionophore A23187- (0.5 and 2.5 microM) stimulated cells SAA1 led to a pronounced dose-dependent increase of TXA2, PGE2, and PGF2alpha. In addition a time-dependent increase of cyclooxygenase metabolites in between 1.5- and 3-fold in the presence of SAA1 was observed; apo A-I, the main HDL-apolipoprotein under non-acute-phase conditions, had no effect. Using sequence-specific anti-human SAA1 peptide (40-63) F(ab)2 fragments we could show that the proposed Ca2+-binding tetrapeptide Gly48-Pro49-Gly50-Gys51 of SAA1 is not responsible for enhanced biosynthesis of cyclooxygenase metabolites. Finally, we could demonstrate that human SAA1 is unable to bind Ca2+-ions, suggesting that SAA1 does not directly enhance eicosanoid biosynthesis via Ca2+ mobilization leading to enhanced phospholipase A2 activity.

Selective induction of the monocyte-attracting chemokines MCP-1 and IP-10 in vesicular stomatitis virus-infected human monocytes.

It is characteristic of viral infections that monocytes/macrophages and lymphocytes infiltrate infected tissue, and neutrophils are absent. CC and non-ELR CXC chemokines predominantly attract mononuclear leukocytes, whereas the ELR motif-expressing CXC chemokines primarily act on neutrophils. To investigate the general role of chemokines in viral diseases, we determined their release and expression patterns after infection of human monocytes with vesicular stomatitis virus (VSV). Human monocytes were productively infected by VSV. Surprisingly, VSV did not induce the release of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6. In contrast, we found a strong induction of the CC chemokine monocyte chemotactic protein-1 (MCP-1) and the non-ELR CXC chemokine interferon-gamma (IFN-gamma) inducible protein-10 (IP-10) by VSV on the gene and protein level. The expression and release of the neutrophil chemoattractants IL-8 and growth-related oncogene-alpha (GRO-alpha) remained unaffected after VSV infection. Our results indicate that the typical monocyte and lymphocyte-dominated leukocyte infiltration of virus-infected tissue is based on a selective induction of mononuclear leukocyte-attracting chemokines.

Role of macrophage cytokines in influenza A virus infections.

Human monocytes and murine macrophages were found to be susceptible to infection by influenza A virus. Although virus replication was low, infection led to cell death which was characterized by an extreme intracellular vacuolization. Most importantly, influenza A virus infection was accompanied by a particular pattern of cytokine release. Whereas IL-1 beta, IL-6 and TNF-alpha production was dependent on exposure to infectious virus, IFN-alpha/beta release was also induced by UV-inactivated virus. Although influenza A virus infection alone induced a substantial cytokine mRNA accumulation, translation into bioactive cytokine protein was rather limited. However, addition of low LPS concentrations was capable of strongly potentiating cytokine release from virus-infected cells. Thus, in a first step, an influenza A virus infection primes mononuclear phagocytes by leading to an accumulation of cytokine mRNA which, in a second step, may be readily translated into bioactive cytokines when triggering signals such as LPS are available. These findings suggest that influenza A virus represents an ultimately fatal macrophage activating factor which, when inducing moderate amounts of cytokines, may be beneficial by mounting an immediate antiviral response, but which may cause adverse effects when cytokine release is highly elevated by bacterial products.

Programmed cell death (apoptosis) in human monocytes infected by influenza A virus.

Although infection of monocytes by influenza A virus primes for a high cytokine release, it also leads to cell death within 20-30 hours. In this brief report, we demonstrate that influenza A virus-induced monocyte killing was due to programmed cell death (apoptosis) and not to necrosis. Morphologically, chromatin condensation and margination occurred and biochemically, an apoptosis-specific internucleosomal DNA fragmentation into multimers of 180 bp ("DNA ladder") was found. Induction of apoptosis and not necrosis in influenza A virus-infected monocytes may serve three purposes: 1. Virus replication is limited, 2. a priming for a high cytokine response is initiated and 3. damaging and inflammation-inducing lysosomal enzymes are held back from monocytes undergoing controlled cell death.

Histopathological changes in enlarged thoracic lymph nodes during the development of silicosis in rats.

Silicosis is primarily a fibrotic lung disease which also affects the draining lymph nodes. In the present study, we examined the lymph nodes of rats from 2 weeks to 52 weeks after an 8-day silica aerosol exposure. Parallel to the typical silicotic changes in the lungs, profound alterations occurred in both posterior mediastinal lymph nodes. The weight of the lymph nodes progressively increased from 3.5-fold to 35-fold at 52 weeks after silica exposure. The weight increase was accompanied by an early increase of T cells and preferentially of CD4+ cells at 2 weeks, which converted into a B cell increase at 6 weeks. Histologically, a leukocyte influx without apparent structural changes was noted at 2 weeks whereas at 6 weeks, germinal centers and T cell regions were disappearing and macrophages accumulated in granuloma-like structures which were randomly scattered throughout the lymphoid tissue. Within the granulomas, macrophages were detected that carried ingested silica particles without apparent signs of degeneration or apoptosis. At 52 weeks after silica exposure, macrophage granulomas persisted without induction of fibrosis in both lymph nodes, and T and B cells were now evenly distributed within the tissue. These data extend our previous findings on lymphocyte and macrophage activation and indicate that the early and marked disorganization of draining lymph node structures may contribute to the immune abnormalities in silicosis.

The lack of receptors for atrial natriuretic peptides on human monocytes prevents a rise of cGMP and induction of tumor necrosis factor-alpha synthesis.

Freshly harvested human monocytes were shown to produce tumor necrosis factor-alpha (TNF-alpha) in response to exogenously added or sodium nitroprusside-generated cGMP. In contrast, atrial natriuretic peptide (ANP) that acts by elevating cGMP in a variety of cells, was incapable of inducing TNF-alpha synthesis. This failure was due to a lack of ANP receptors and thus, to the inability of ANP to raise cGMP in human monocytes.

The receptor for interleukin 1 in plasma membranes of the human leukemia cell K 562: biological and biochemical characterization.

Murine interleukin 1 (IL 1) inhibited concentration dependently the proliferation of murine T cell lymphomas and the human leukemic cell line K 562. The cytostatic action of IL 1 was not associated with cytotoxicity and appeared to be irreversible. Changes in the expression of surface antigens, like a rapid decrease of transferrin receptors or, more delayed, an increase in HLA-A, B, C antigen density suggested that a differentiation step was induced by IL 1. This effect of IL 1 was a direct one and most likely mediated by a specific receptor molecule. In order to characterize the receptor for IL 1, highly purified plasma membranes from K 562 were incubated with murine IL 1, and the phosphorylation pattern of plasma membrane proteins was investigated by the addition of radiolabeled ATP. At 0 degree C, IL 1 induced the specific phosphorylation of a 41 kDa membrane protein in a time- and concentration-dependent manner. Analysis of the phosphoamino acid composition revealed that IL 1 induced specifically the phosphorylation of tyrosine residues of the 41 kDa protein. Crosslinking experiments proved that the 41 kDa protein had an IL 1 binding site, strongly suggesting that the 41 kDa protein was the receptor for IL 1 itself. Affinity labeling with an ATP-analogue showed that this protein possessed an ATP binding and cleaving site. We conclude from this that the receptor for IL 1 in the plasma membranes of K 562 is a transmembranous protein of 41 kDa, which possesses a tyrosine specific protein kinase activity with an autophosphorylating capacity.

Effects of cyclosporin A on functions of specific murine T cell clones: inhibition of proliferation, lymphokine secretion and cytotoxicity.

Allospecific T lymphocyte clones with different functions were generated from spleen cells of C 57/Bl6 mice following sensitization in vitro by a one-way mixed lymphocyte culture (MLC) with irradiated DBA/2 spleen cells. The clones were propagated in vitro in the presence of interleukin 2 (IL 2) and restimulation with stimulator cells. In these clones Cyclosporin A (CSA) was tested for its suppressive effect on different T lymphocyte functions. The antigen-dependent proliferation of a helper clone (HTL) was totally inhibited by 50 ng/ml CSA. Proliferation induced by simultaneous administration of antigen and IL 2 was partially suppressed in all helper and cytotoxic clones (CTL). The IL 2-driven proliferation in the absence of antigen was also suppressed between 25-70% by the immunosuppressive drug. Secretion of macrophage activating factor (MAF) and interferon (IFN) by HTL and CTL in response to antigen or mitogen was reduced dose dependently by CSA. Concentrations of 50 ng/ml CSA diminished lymphokine secretion to approximately 10% of controls, also when excess IL 2 was present. Cytotoxicity, previously described to be insensitive to the drug, could be suppressed by 50 ng/ml CSA to a various extent, from 40-70%, in different cytotoxic clones when the effector cells were preincubated with CSA for 1 h or more. Conclusively, the data suggest that CSA interferes generally with the activation of T lymphocyte clones.

In vivo activation of macrophages by T cell-derived lymphokines: killing of tumor cells and schistosomula of Schistosoma mansoni.

We investigated the role of T cell-derived lymphokines for macrophage activation in vivo. We show for the first time that macrophages from casein-pretreated mice can be primed in vivo by intraperitoneal injection of immune interferon (IFN-gamma) and can be triggered by lipopolysaccharide (LPS) in vitro to kill schistosomula of S. mansoni. Similar results were obtained for the activation of tumoricidal macrophages. Injection of casein-pretreated mice with concanavalin A (Con A)-induced supernatant of a long-term T cell clone containing IFN-gamma and macrophage cytotoxicity inducing factor 2 (MCIF2), however, induced macrophage activation in vivo without further addition of LPS in vitro. These experiments show that macrophages can be activated by lymphokines in vivo. In addition, the data suggest that a combination of IFN-gamma with MCIF2 might be more effective than IFN-gamma alone. These data may be relevant for the strategy of treating cancer and infectious diseases with lymphokines.

Inhibition of LPS-induced tumor necrosis factor-alpha production by colchicine and other microtubule disrupting drugs.

Colchicine has been shown to act as an antiinflammatory agent. In this study, we examined whether colchicine and other microtubule-depolymerizing drugs affected the production of TNF-alpha. When rat peritoneal macrophages were stimulated by LPS, addition of colchicine, vincristine, vinblastine or nocodazole was found to inhibit TNF-alpha release in a concentration-dependent manner. Suppression of TNF-alpha release was not due to interference with secretion as the cytokine did not accumulate intracellularly following colchicine treatment. Colchicine markedly enhanced PGE2 release from LPS-stimulated macrophages. However, addition of the cyclooxygenase inhibitor indomethacin only partially reversed the suppressive effect of colchicine on TNF-alpha production. Colchicine caused a strong reduction of LPS-induced TNF-alpha mRNA accumulation, suggesting that a pretranslational effect may represent the primary mechanism by which colchicine reduced TNF-alpha production. These observations could have clinical relevance in ameliorating undesirable effects due to excessive TNF-alpha production, for example following LPS stimulation of monocytes/macrophages in gram-negative sepsis. Furthermore, these drugs may provide useful tools to study the apparent involvement of the microtubular system in cytokine gene expression and cytokine production.

Suppression of LPS-induced tumor necrosis factor-alpha gene expression by microtubule disrupting agents.

Microtubule disrupting agents such as colchicine have been shown to reduce TNF-alpha production in macrophages. To examine molecular mechanisms underlying the action of colchicine, TNF-alpha gene expression was studied in the murine macrophage cell line PU5-1.8. An LPS stimulation caused an intense up-regulation of TNF-alpha gene expression which was followed by a high TNF-alpha protein production. Simultaneous addition of colchicine (10 microM) suppressed LPS-induced TNF-alpha mRNA accumulation by one-third and TNF-alpha protein release by two-thirds. This effect was shared by vinblastine and vincristine, chemically different agents that also disrupt microtubule polymerization. For full suppressive activity on TNF-alpha gene expression, colchicine had to be present for 3 h in LPS-stimulated macrophage cultures. With nuclear run-on transcription experiments we could demonstrate that colchicine primarily inhibited de novo gene transcription and did not accelerate degradation of TNF-alpha mRNA in actinomycin D-treated macrophages. Thus, the well-known antiinflammatory action of microtubule depolymerizing agents may be largely due to a reduced TNF-alpha gene expression.

Expression of transcription factor genes after influenza A virus infection.

Infection of human monocytes with influenza A virus induces a broad range of proinflammatory cytokines and mononuclear cell attracting chemokines before the infected cells undergo apoptosis. The underlying mechanisms by which the corresponding genes are transcriptionally initiated after virus infection are still poorly understood. Activation of NF-kappa B seems to play an important role in the regulation of many proinflammatory cytokine genes, but cannot be the only mechanism, since several cytokine genes lack respective binding sites in their promoter regions. Therefore, we additionally investigated other transcription factors of possible importance such as CREB, CTF, OTF-1, and OTF-2. To explore long-term regulatory mechanisms, we investigated the induction of transcription factors on the gene expression level which may be important to substitute for metabolized transcription factor proteins after their activation. We identified a cell-type-specific differential response: CREB, CTF, OTF-1, OFT-2, and NF-kappa B genes were strongly induced 1 to 4 hours after influenza A virus infection in the monocytic cell line Mono Mac 6, while in freshly prepared human monocytes no significant changes were detected. In infected monocytes, which die by apoptosis, the expression of CREB, CTF, and OTF-2 was rather suppressed 8 hours after infection. In conclusion, the long-term regulation of transcription factor gene expression in non-proliferating cells seems to be of minor importance after influenza infection since in apoptosisprone cells an immediate availability of transcription factor proteins is required.

Suppression of T lymphocyte proliferation during lymphoma growth in mice: role of PGE2-producing suppressor macrophages.

The suppressed T lymphocyte response occurring during lymphoma growth in mice was largely due to the generation of suppressor macrophages which released high amounts of prostaglandin E2 (PGE2). Based on these findings, the role of macrophage-derived PGE2 as a regulating and potentially immunosuppressive agent of the immune response is discussed.

Enhancement of tumor necrosis factor-alpha gene expression by low doses of prostaglandin E2 and cyclic GMP.

Macrophage-derived PGE2 is usually considered to be a down-regulator of TNF-alpha production. However, we recently demonstrated that PGE2 may display dual activities in that low concentrations stimulated whereas higher doses suppressed TNF-alpha synthesis in resident peritoneal macrophages. To examine the underlying molecular mechanisms, we studied TNF-alpha gene expression in rat peritoneal macrophages and the murine PU5-1.8 macrophage line. In both macrophage types, PGE2 enhanced TNF-alpha gene transcription and production at an optimal concentration of 1 ng/ml. Furthermore, evidence was obtained that PGE2 may stimulate TNF-alpha mRNA accumulation via a rise of the intracellular messenger cGMP. Both, exogenously added as well as endogenously, by sodium nitroprusside generated cGMP were found to enhance TNF-alpha gene expression and production. These findings lend further support to the concept that cGMP may represent one of the positive signals for TNF-alpha synthesis.

Interleukin 1 as a tumor cytostatic mediator released from tumor ascites-treated macrophages.

Peritoneal macrophages from DBA/2 mice, elicited by injection of Corynebacterium parvum (C.p.), were in vitro activated to Eb tumor cytostasis by incubation with tumor-induced ascites that was harvested 7 days after intraperitoneal Eb injection. The active cytostasis-mediating compound was found to be interleukin 1 (IL 1). When tumor ascites was fractionated according to molecular weight size, the most active IL 1-inducing fraction was found to comprise molecules of greater than 100,000 daltons. The data show that tumor-bearing hosts are capable of producing compounds that induce a high IL 1 secretion which may enable macrophages to mount an antiproliferative effect against tumor cells.