In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha

Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-alpha, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-alpha and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-alpha or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-alpha or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-alpha and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-alpha or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-alpha and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.     

Compartmentalized roles for leukocytic adhesion molecules in lung inflammatory injury

By using the model of acute injury caused by intrapulmonary deposition of IgG immune complexes, blocking mAb to CD11a, CD11b, L-selectin, and intercellular adhesion molecule-1 (ICAM-1) were administered either i.v. or intratracheally (i.t.). The effects of these interventions were assessed according to lung injury, lung content of myeloperoxidase (MPO), TNF-alpha, and cellular content in bronchoalveolar lavage (BAL) fluids, and up-regulation of pulmonary vascular ICAM-1. In animals treated i.v. with Abs to CD11a, L-selectin, or ICAM-1 lung injury was significantly attenuated in parallel with reduced lung content of MPO. Under similar conditions, treatment with anti-CD11b had no effect. However, when the same mAb were administered i.t., anti-CD11a and anti-L-selectin were without protective effects, whereas i.t. administered anti-CD11b and anti-ICAM-1 were each highly protective. The protective effects of anti-CD11b were related to profound reductions in BAL levels of TNF-alpha, pulmonary vascular up-regulation of ICAM-1, and lung content of MPO. The protective effects of i.t.-administered anti-ICAM-1 were not associated with reduced BAL levels of TNF-alpha. Protective effects of mAb were also reflected in reductions of retrievable neutrophils in BAL fluids. mAb to rat CD11b and CD18 but not to rat CD11a suppressed in vitro production of TNF-alpha by immune complex-stimulated rat alveolar macrophages. The mAb did not reduce NO2-/NO3- generation in stimulated macrophages but all mAb (except anti-ICAM-1) reduced O2- responses in macrophages. These data suggest a compartmentalized role for adhesion molecules in lung inflammatory injury after intraalveolar deposition of IgG immune complexes, with CD11a, L-selectin, and ICAM-1 being important in the vascular compartment for neutrophil recruitment, whereas in the alveolar compartment CD11b and ICAM-1 (but not CD11a and L-selectin) seem to play key roles.     

Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.     

Superoxide mediates cigarette smoke-induced infiltration of neutrophils into the airways through nuclear factor-kappaB activation and IL-8 mRNA expression in guinea pigs in vivo

We examined the hypothesis that superoxide mediates infiltration of neutrophils to the airways through nuclear factor (NF)-kappaB and interleukin-8 (IL-8) after acute exposure to cigarette smoke (CS) in vivo. Male Hartley strain guinea pigs were exposed to air or 20 puffs of CS and killed 5 h after the exposure. The differential cell count of bronchoalveolar lavage fluid and specific myeloperoxidase enzyme assay demonstrated that acute exposure to CS caused neutrophil accumulation to the airways and parenchyma, respectively. Acute exposure to CS increased DNA-binding activity of NF-kappaB in the lung. Acute exposure to CS also increased IL-8 messenger RNA (mRNA) expression in the lung. Pretreatment of guinea pigs with recombinant human superoxide dismutase (rhSOD) aerosols reduced the CS-induced neutrophil accumulation to the airways. Both activation of NF-kappaB and increased IL-8 mRNA expression were also inhibited by the pretreatment of rhSOD aerosols. Strong immunoreactivities for p65 and p50 were detected in the nuclei of alveolar macrophages after acute exposure to CS. The signal for IL-8 mRNA expression was demonstrated in the alveolar space after acute exposure to CS. Neither significant immunoreactivities for p65 and p50 nor IL-8 mRNA signals were observed in airway epithelium. These observations suggest that acute exposure to CS initiates superoxide-dependent mechanism that, through NF-kappaB activation and IL-8 mRNA expression, produces infiltration of neutrophils to the airways in vivo. It was also suggested that the alveolar macrophage is one potential source of NF-kappaB activation and IL-8 mRNA expression after acute exposure to CS.     

TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages

Macrophages are present in inflammatory tissue sites where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of macrophages to participate in such matrix destruction, we studied the effects of three cytokines present in inflammatory tissue sites, TNF-alpha, IL-1beta, and IFN-gamma, on the production of three matrix-degrading metalloproteinases, interstitial collagenase, stromelysin, and 92-kDa gelatinase, as well as their natural inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases number 1), by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of interstitial collagenase and stromelysin by these cells was minimal, and was not influenced by the cytokines. In contrast, the cells secreted substantial basal amounts of 92-kDa gelatinase, the secretion of which was stimulated (2- to 15-fold; on average 5-fold) by both TNF-alpha and IL-1beta, while the production of TIMP-1 was unaffected. IFN-gamma suppressed the production of the 92-kDa gelatinase induced by TNF-alpha- and IL-1beta. TNF-alpha and IL-1beta regulated the expression of 92-kDa gelatinase by monocyte-derived macrophages at the pretranslational level. The results show that expression of 92-kDa gelatinase, but not its natural inhibitor TIMP-1, by human tissue-type macrophages is selectively up-regulated by proinflammatory cytokines; which suggests that these cells, when actually present in an inflammatory environment, will actively participate in the destruction of the extracellular matrix.     

Acid sphingomyelinase-derived ceramide is not required for inflammatory cytokine signalling in murine macrophages

Sphingomyelin hydrolysis is induced in myeloid cell-lines by tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1beta), and interferon gamma (IFN-gamma). Ceramide, a product of sphingomyelin hydrolysis, recapitulates many of the cellular responses elicited by these cytokines, and this has lead to the hypothesis that ceramide is a second messenger of cytokine signalling. Sphingomyelin hydrolysis is catalysed by an acid spingomyelinase (ASMase) and one or more neutral sphingomyelinases (NSMase); both ASMase and NSMase are activated during cytokine signalling. In the present study, the contribution of ASMase to TNF-alpha, IL-1beta, and IFN-gamma signalling in murine macrophages was addressed. Cytokine-induced responses were compared in macrophages derived from the bone marrow of AMSase null and wild-type mice. Specifically, TNF-alpha-and IFN-gamma-induced nitric oxide production and TNF-alpha- and IL-1beta-induced expression of the alpha-chemokine, KC, were intact in ASMase null macrophages. Furthermore, TNF-alpha induction of p42/p44 ERK and p38-MAPK phosphorylation, c-jun kinase activation, and IkappaBalpha degradation were normal. Also normal in ASMase null macrophages was TNF-alpha-, IL-1beta- and IFN-gamma-induced expression of a panel of early response genes. It is concluded that ASMase is non-essential for the inflammatory signals activated in murine macrophages by TNF-alpha, IL-1beta and IFN-gamma.     

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The effect of nitric oxide on the lipopolysaccharide (LPS)-induced cytokine production by alveolar macrophages was studied. When alveolar macrophages were cultured, substantial amounts of interleukin-1(IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha(TNF-alpha), and nitric oxide are produced upon stimulation with LPS. Inhibition of the nitric oxide production by the L-arginine analogue N(G)-monomethyl-L-arginine (NMMA), resulted in an increase of IL-1(beta) and IL-6, whereas the TNF-alpha concentrations remained unchanged, suggesting specific inhibitory effects of nitric oxide on the LPS-stimulated cytokine production by alveolar macrophages. The observed cytokine-modulation properties of nitric oxide did not result from cytotoxic actions of the oxidation of L-arginine on macrophages, since nitric oxide synthesis did not affect the viability of the alveolar macrophages. Conversely the nitric oxide donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated alveolar macrophages in which endogenous nitric oxide production was blocked. The results indicate that nitric oxide can affect the LPS-induced IL-1beta and IL-6 secretion by alveolar macrophages in an autoregulatory way and are discussed in view of the important physiologic consequences this autoregulation by nitric acid oxide may have.     

The neurotoxin MPTP increases calbindin-D28k levels in mouse midbrain dopaminergic neurons

Interleukin (IL)-2-induced microvascular lung injury is an experimental paradigm commonly used to investigate the pathogenesis of the adult respiratory distress syndrome. Since tumor necrosis factor-alpha (TNF-alpha) is known to induce such an injury in vivo and since TNF-alpha is involved in other models of lung injury, we postulated that it might also mediate pulmonary toxicity after IL-2 administration. The present study tested this hypothesis by evaluating the effect of TNF-alpha inhibition on IL-2-induced lung injury in the rat. Recombinant human IL-2 (10(6) U IV per rat, n = 6) elevated lung water, myeloperoxidase activity, and protein accumulation in bronchoalveolar lavage fluid and induced tissue hypoxia. Also, IL-2 enhanced lung tissue TNF-alpha mRNA and peptide (1543 +/- 496 pg/g lung wet weight) localized to alveolar macrophages by in situ hybridization. In marked contrast, IL-2 failed to affect serum TNF-alpha, which remained at undetectable levels. Pretreatment with anti-TNF-alpha monoclonal antibody (25 mg/kg IV, n = 7) or the TNF-alpha synthesis inhibitor rolipram (200 micrograms/kg IV, n = 7) attenuated lung injury and reverted tissue hypoxia. Furthermore, TNF-alpha inhibition prevented the upregulation of lung tissue IL-1 beta, IL-6, cytokine-induced neutrophil chemoattractant, and E-selectin (ELAM-1) but not intercellular adhesion molecule-1 mRNAs in response to IL-2. These data imply that locally produced TNF-alpha mediates IL-2-induced lung inflammation and tissue injury and point to the potential utilization of TNF-alpha inhibitors in treating the pulmonary toxicity of IL-2 immunotherapy.     

Decision tree in hematologic diseases: for a rationalization of histologic, phenotypic and genotypic diagnosis

The regulatory role of soluble cytokines in innate cellular immune responses induced by Pneumocystis carinii was assessed in vitro in direct comparison to induction by Listeria monocytogenes. This report shows that P. carinii organisms, as well as L. monocytogenes, stimulated in whole spleen cell cultures of SCID mice the release of IFN-gamma, TNF-alpha/beta, IL-10, IL-12, and iNO. This response was independent of functional T cells. Both macrophages (M phi) and natural killer (NK) cells were necessary for either microorganism to induce release of these cytokines. Cocultures of purified M phi--including alveolar M phi--and purified NK cells indicated that no other cell population was necessarily involved. Microbial induction of NK cell-derived IFN-gamma has been reported to be mediated by the combined effects of TNF-alpha and IL-12 released by M phi upon adequate microbial stimulation. Interestingly, only L. monocytogenes, but not P. carinii organisms could directly induce detectable amounts of TNF-alpha/beta, IL-12, or iNO in purified M phi cultures. In dose-response experiments, release of IFN-gamma, TNF-alpha/beta, and iNO was reduced at high relative concentrations of either microorganism. This high-dose suppression was at least partially controlled by M phi-produced IL-10. Our data show that, P. carinii potently induces activating and inhibitory innate cellular immune response mechanisms and indicate that the initial step of macrophage-mediated NK cell activation might also involve other pathways than those described to date.     

Acute lung injury: the role of cytokines in the elicitation of neutrophils

Cytokine networks between immune and nonimmune cells of the alveolar-capillary membrane are necessary for cellular communication during pulmonary inflammation. The subsequent events of these cellular/humoral interactions are pivotal to the initiation and propagation of the inflammatory response leading to pulmonary injury. The studies cited in this paper underscore the interrelationship of early response cytokines, adhesion molecules, and the chemokine IL-8 that orchestrate the recruitment of neutrophils into the lung. The paradigm for neutrophil extravasation is likely operative in the microvasculature of the lung, and consists of four or more steps (Figure 3). First, acute lung injury results in the activation of microvascular endothelium in response to the local generation of TNF or IL-1, leading to expression of endothelial cell-derived E- and P-selectins and ICAM-1. The constitutive presence of neutrophil-derived L-selectin allows for the initial adhesive interaction of neutrophils with endothelial cell selectins leading to the "rolling" effect. Second, generation of IL-8 leads to the activation of neutrophils in the vascular compartment and expression of beta 2 integrins, while L-selectin is concomitantly shed. Third, the interaction of the neutrophil beta 2 integrin with its receptor/ligand, ICAM-1, results in the rapid arrest of neutrophils on the endothelium. Fourth, the subsequent events leading to neutrophil extravasation beyond the vascular compartment are dependent upon a combination of haplotaxis (migration in response to an insoluble gradient), the continued expression of beta 2 integrins on neutrophils and ICAM-1 on nonimmune cells, and the maintenance of a neutrophil specific (IL-8) chemotactic gradient. The participation of IL-8 and potentially other C-X-C chemokines in the inflammatory response appears to be critical for the orchestration of the directed migration of inflammatory leukocytes into the lung. After arriving in the lung, these activated leukocytes can respond to noxious stimuli or induce pulmonary injury through the release of reactive oxygen metabolites, proteolytic enzymes, and additional cytokines. Our current knowledge and future investigations regarding the mechanisms involved in neutrophil elicitation may allow us to employ clinical interventional strategies that will attenuate neutrophil-dependent acute lung injury, such as ARDS.     

Production of tumor necrosis factor alpha and interleukin-6 by alveolar macrophages from patients with rheumatoid arthritis and interstitial pulmonary disease

The aim of this study was to measure the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by alveolar macrophages in patients with rheumatoid arthritis and interstitial lung disease (ILD). Rheumatoid arthritis patients diagnosed by ACR criteria (n = 8) with associated ILD documented by pulmonary function tests and high resolution computerized tomography scanning, and 12 healthy volunteers (6 smokers and 6 nonsmokers). We determined the spontaneous and induced production of bacterial lipopolysaccharides (LSP), TNF-alpha and IL-6 by alveolar macrophages obtained by bronchoalveolar lavage. The macrophages were isolated by Ficoll-Hypaque gradient centrifugation and plastic adherence and cultured in serum-containing medium (low endotoxin) in the presence and absence of LPS (100 ng/ml). TNF-alpha and IL-6 levels contents were determined in supernatants by ELISA. In the patient group both spontaneous and induced production of TNF-alpha were significantly higher than in controls (p < 0.01). Macrophages stimulated with LPS in patients with rheumatoid arthritis and ILD also released greater amounts of IL-6 than did those of the healthy controls. IL-6 spontaneous and induced production was significantly lower in smokers than in nonsmokers. TNF-alpha and IL-6 production in patients with rheumatoid arthritis and ILD, studied in bronchoalveolar lavage specimens reveals that alveolar macrophages are hyperreactive in these patients, who are possibly sensitized as a consequence of the inflammatory lung process inherent to the disease. Further study is needed to define the pathogenic role of these mediators.     

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.     

Involvement of the IL-2 receptor gamma-chain (gammac) in the control by IL-4 of human monocyte and macrophage proinflammatory mediator production

IL-4 has potent anti-inflammatory properties on monocytes and suppresses both IL-1beta and TNF-alpha production. Well-characterized components of the IL-4 receptor on monocytes include the 140-kDa alpha-chain and the IL-2R gamma-chain, gammac, which normally dimerize 1:1 for signaling from the receptor. However, mRNA levels for gammac were very low in 7-day-cultured monocytes. As mRNA levels for gammac declined with culture, so too did the ability of IL-4 to down-regulate LPS-induced TNF-alpha production. In contrast, IL-4 consistently down-regulated IL-1beta production by cultured monocytes. Immunoprecipitation and Western blot analyses demonstrated that 7-day-cultured monocytes do not express the functionally active 64-kDa gammac protein. This was associated with decreased STAT6 activation by IL-4. Studies with Abs to gammac and an IL-4 mutant that is unable to bind to gammac showed that IL-4 can suppress IL-1beta but not TNF-alpha production by LPS-stimulated monocytes in the presence of little or no functioning gammac. IL-4 also suppressed IL-1beta but not TNF-alpha production by Mono Mac 6 cells, which express minimal levels of gammac. For gammac-expressing LPS/PMA-activated U937 cells, IL-4 decreased both TNF-alpha and IL-1beta production. These results suggest that functional gammac is not present on in vitro-derived macrophages, and that while some anti-inflammatory responses to IL-4 are lost with this down-regulation of functional gammac, others are retained. We conclude that different functional responses to IL-4 by human monocytes and macrophages are regulated by different IL-4 receptor configurations.     

Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma

A 56-year-old female with rheumatoid arthritis was admitted because of bilateral hip pain. In a few months of her hospitalization, a relatively abrupt renal dysfunction was emerged besides complement breakdown, and renal biopsy revealed crescentic glomerulonephritis. Immunofluorescence study showed peripheral granular deposits of IgG, IgM, and C3 in the glomeruli. Cresents were predominantly composed of macrophages and glomerular epithelial cells. Amyloid nephropathy, renal vasuculitis, and association of other collagen vascular diseases were negligible for the causative factor. It was suggested that immune complexes were formed in the glomeruli, in which both humoral and cellular immune responses were to be induced, that brought cescents formation in the lesions. Crescentic glomerulonephritis in patients with rheumatoid arthritis is rare and a possible pathogenetic mechanisms involved in the development of renal dysfunction are discussed with the special reference to immune complex-induced inflammation.