Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

Background

Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM₁₀) in vitro increase their production of inflammatory mediators and that supernatants from PM₁₀-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.

Methods

The present study concerns co-culture of AM and HBEC exposed to PM₁₀ (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.

Results

AM/HBEC co-cultures exposed to 100 μg/ml of PM₁₀ for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM₁₀-induced increase in co-culture mRNA expression.

Conclusion

We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM₁₀-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.     

Evaluation of TNF-alpha and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-alpha/anti-CD4 therapy

We have evaluated the effects of anti-TNF-alpha, anti-IL-1, and combined anti-TNF-alpha/anti-CD4 therapy in collagen-induced arthritis. Blockade of TNF-alpha or IL-1 before disease onset delayed, but did not prevent, the induction of arthritis. When treatment was initiated after onset of arthritis, anti-TNF-alpha, anti-IL-1beta, and anti-IL-1R (which blocks IL-1alpha and IL-1beta) were all found to be effective in reducing the severity of arthritis, with anti-IL-1R and anti-IL-1beta showing greater efficacy than anti-TNF-alpha. Anti-IL-1beta was equally as effective as anti-IL-1R, indicating that IL-1beta plays a more prominent role than IL-1alpha in collagen-induced arthritis. An additive effect was observed between anti-TNF-alpha and anti-IL-1R in the prevention of joint erosion and in normalization of the levels of serum amyloid P. Combined anti-TNF-alpha/anti-CD4 therapy also caused normalization of serum amyloid P levels. The therapeutic effect of anti-TNF-alpha plus anti-CD4 was comparable to that of anti-TNF-alpha plus anti-IL-1R, suggesting that combined anti-TNF-alpha/anti-CD4 therapy prevents both TNF-alpha- and IL-1-mediated pathology. Anti-TNF-alpha treatment reduced IL-1beta expression in the joint and, conversely, anti-IL-1beta treatment reduced TNF-alpha expression. Combined anti-TNF-alpha/anti-CD4 treatment almost completely blocked the expression of IL-1beta, thereby confirming the ability of this form of combination therapy to prevent IL-1ss-mediated pathology.     

HMGB1 develops enhanced proinflammatory activity by binding to cytokines

High mobility group box 1 protein (HMGB1), originally characterized as a nuclear DNA-binding protein, has also been described to have an extracellular role when it is involved in cellular activation and proinflammatory responses. In this study, FLAG-tagged HMGB1 was inducibly expressed in the presence of culture media with or without added IL-1beta, IFN-gamma, or TNF-alpha. HMGB1 purified from cells grown in culture media alone only minimally increased cytokine production by MH-S macrophages and had no effect on murine neutrophils. In contrast, HMGB1 isolated from cells cultured in the presence of IL-1beta, IFN-gamma, and TNF-alpha had enhanced proinflammatory activity, resulting in increased production of MIP-2 and TNF-alpha by exposed cells. IL-1beta was bound to HMGB1 isolated from cells cultured with this cytokine, and purified HMGB1 incubated with recombinant IL-1beta acquired proinflammatory activity. Addition of anti-IL-1beta Abs or the IL-1 receptor antagonist to cell cultures blocked the proinflammatory activity of HMGB1 purified from IL-1beta-exposed cells, indicating that such activity was dependent on interaction with the IL-1 receptor. These results demonstrate that HMGB1 acquires proinflammatory activity through binding to proinflammatory mediators, such as IL-1beta.     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

The effects of PM10 particles and oxidative stress on macrophages and lung epithelial cells: modulating effects of calcium-signaling antagonists

We have previously examined the ability of air pollution particles (PM(10)) to promote release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) from human peripheral blood mononuclear cells and demonstrated a role for calcium as a signaling molecule in this process. We have now studied the ability of oxidative stress induced by a synthetic oxidant tert-butyl hydroperoxide (tBHP) to induce TNF-alpha production via calcium signaling in the mouse macrophage cell line (J774). The oxidant tBHP significantly increased intracellular calcium and the release of TNF-alpha in J774 cells, an effect that was reduced to control levels by inhibition of calcium signaling with verapamil, BAPTA-AM, and W-7. This study also investigated interactions between PM(10)-treated macrophages and epithelial cells by using conditioned medium (CM) from PM(10)-treated mononuclear cells to stimulate the release of the neutrophil chemoattractant chemokine IL-8 from A549 lung epithelial cells. TNF-alpha protein release was demonstrated in human mononuclear cells after PM(10) treatment, an effect that was inhibited by calcium antagonists. Treatment of A549 cells with monocyte/PM(10) CM produced increased IL-8 release that was reduced with CM from monocyte/PM(10)/calcium antagonist treatments. The expression of ICAM-1 was increased after incubation with CM from monocyte/PM(10) treatment, and this increase was prevented by treatment with CM from monocyte/PM(10)/calcium antagonist. These data demonstrate a link between oxidative stress, calcium, and inflammatory mediator production in macrophages and lung epithelial cells.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta

The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Cytokine network regulating terminal maturation of human bone marrow B cells capable of spontaneous and high rate Ig secretion in vitro.

Human bone marrow (BM) B cells capable of spontaneous and high rate Ig secretion for 14 days in vitro have been described previously. We have shown recently that Ig secretion by these BM cells depends on stromal adherent BM cell-derived factors identified as IL-6 and fibronectin. Our report shows that the endogenous generation of IL-1 beta and TNF-alpha in serum-containing cultures of BM mononuclear cells (BMMC) is also involved in the control of Ig-secreting cells, because their blockade with specific antibodies markedly reduced Ig production. Further experiments revealed that IL-1 beta and TNF-alpha acted by regulating IL-6 production, as can be deduced from the following findings: 1) the inhibition of Ig secretion caused by either anti-IL-1 beta or anti-TNF-alpha antibodies could be reversed by exogenous IL-6; 2) the addition of either of these antibodies inhibited endogenous IL-6 production in BMMC cultures; 3) IL-1 beta plus TNF-alpha, but neither one alone, restored complete IL-6 and Ig production by BMMC in serum-free cultures. Moreover, adherent, but not nonadherent, BM cells were responsible for endogenous IL-1 beta and TNF-alpha secretion. Finally, IL-1 beta plus TNF-alpha induced the production of IL-6, but not of Ig, by adherent BM cells. Neither IL-6 nor Ig production was induced by adding this cytokine combination to nonadherent BM cell cultures, despite the fact that this fraction contained all the Ig-secreting cells. However, the addition of IL-6 restored Ig secretion in this cell fraction. These results suggest that IL-1 beta and TNF-alpha produced by adherent BM cells synergistically induce early IL-6 generation, which, in turn, drives BM B cell producers into the high rate Ig-secreting state.     

Cutting edge: IL-1beta mediates the proangiogenic activity of osteopontin-activated human monocytes

Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1beta up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-alpha, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1beta mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1beta.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.     

Bronchial epithelial cell growth regulation in fibroblast cocultures: the role of hepatocyte growth factor

Proliferation of bronchial epithelial cells is an important biological process in physiological conditions and various lung diseases. The objective of this study was to determine how bronchial fibroblasts influence bronchial epithelial cell proliferation. The proliferative activity in cocultures was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct cells counts. Concentration of cytokines was measured in cell culture supernatants by means of ELISA. In primary cell cocultures, fibroblasts or fibroblast-conditioned medium enhanced 1.85-fold the proliferation of primary bronchial epithelial cells (P < 0.02) compared with bronchial epithelial cells cultured alone. The proliferative activity in cocultures and in fibroblast-conditioned medium was reduced by neutralizing antibody to hepatocyte growth factor (HGF) and HGF receptor c-met. Neutralizing antibodies to FGF-7 and IGF-1 had no effect. Treatment of fibroblast-epithelial cocultures with anti-IL-6 and anti-TNF-alpha neutralizing antibodies and with indomethacin decreased production of HGF. These results indicate that cytokines and PGE(2) may indirectly mediate epithelial cell proliferation via the regulation of HGF in bronchial stromal cells and that HGF plays a crucial role in proinflammatory cytokine-induced proliferation in the experimental system studied.