Role of ICAM-1 in the aggregation and adhesion of human alveolar macrophages in response to TNF-alpha and INF-gamma

Intracellular adhesion molecule-1 (ICAM-1)-mediated cell-cell adhesion is thought to play an important role at sites of inflammation. Recent evidence suggests that ICAM-1 surface expression on alveolar macrophages is increased in pulmonary sarcoidosis and that inflammatory granuloma formation is characterized by the aggregation of macrophages. The present study shows that ICAM-1 expression is significantly elevated on alveolar macrophages from patients with sarcoidosis in response to tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma) compared with healthy controls. Aggregation and adhesion were significantly increased in alveolar macrophages treated with TNF-alpha and INF-gamma, and significantly inhibited in those pretreated with a monoclonal antibody to ICAM-1. Similarly, aggregation and adhesion were inhibited in macrophages treated with heparin, which then exhibited a wide range of biological activities relevant to inflammation. These results suggested that the surface expression of ICAM-1 on alveolar macrophages in response to TNF-alpha and INF-gamma is important in mediating aggregation and adhesion. Additionally, heparin may be useful for developing novel therapeutic agents for fibrotic lung disease.     

INF-gamma rearranges membrane topography of MHC-I and ICAM-1 in colon carcinoma cells.

Flow-cytometric fluorescence energy transfer (FCET) measurements between fluorescently labeled cell surface MHC-I and ICAM-1 molecules indicated similar receptor patterns in the plasma membrane of interferon-gamma (INF-gamma)-treated colon carcinoma cells as those observed earlier at the surface of lymphoid cells. INF-gamma activation significantly increased the density of MHC-I and ICAM-1 proteins in the membrane. This increase in receptor density was accompanied by decreased proximity level of the homo-associated MHC-I receptors. Hetero-association of MHC-I and ICAM-1 molecules was increased by INF-gamma treatment. INF-gamma changed neither hetero- nor homo-association of transferrin receptors. By staining the sphingomyelin/cholesterol-enriched lipid microdomains with fluorescently labeled cholera toxin B subunit, we found an increase in the amount of lipid-raft associated G(M1)-gangliosides due to INF-gamma treatment. Confocal microscopic results and FCET measurements show that MHC-I and ICAM-1 are components of G(M1)-ganglioside containing lipid-rafts and also support an increase in the size of these lipid-rafts upon INF-gamma treatment.     

The effects of silica dust and alveolar macrophages on lung fibroblasts grown in vitro

The effect of silica (min-u-sil) on lung fibroblasts, with and without the mediation of alveolar macrophages, has been studied by measuring DNA, protein, collagen, lysosomal enzymes and secreted glycosaminoglycans. Intact macrophages were found to stimulate collagen production whether they had first been pretreated with silica or not. The dust has a direct effect on fibroblasts, an effect dependent on silica concentration and the stage of fibroblast growth. The possible relationships of the above effects to both fibrosis and emphysema are discussed.     

Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro

Lipopolysaccharide (LPS)-induced lung inflammation is known to increase pulmonary intercellular adhesion molecule-1 (ICAM-1) expression. In the present study, L2 cells, a cell line of alveolar epithelial cells, were stimulated with LPS, and ICAM-1 expression was studied. ICAM-1 protein on L2 cells peaked at 6 (38% increase; P < 0.01) and 10 (48% increase; P < 0.001) h after stimulation with Escherichia coli and Pseudomonas aeruginosa LPS, respectively. ICAM-1 mRNA expression was markedly increased, with a peak at 2-4 (E. coli) and 4-6 (P. aeruginosa) h. Adherence assays of neutrophils to LPS-stimulated L2 cells showed a threefold increase in adherence (P < 0.001). Pretreatment of the neutrophils with anti-lymphocyte function-associated antigen-1 and anti-Mac-1 antibodies reduced adherence by 54% (P < 0.001). Analysis of immunofluorescence staining for ICAM-1 showed an exclusive apical expression of ICAM-1. These results indicate that LPS upregulates functional active ICAM-1 on the apical part of the membrane in rat pneumocytes.     

Airway contribution to alveolar nitric oxide in healthy subjects and stable asthma patients

Alveolar nitric oxide (NO) concentration (Fa(NO)), increasingly considered in asthma, is currently interpreted as a reflection of NO production in the alveoli. Recent modeling studies showed that axial molecular diffusion brings NO molecules from the airways back into the alveolar compartment during exhalation (backdiffusion) and contributes to Fa(NO). Our objectives in this study were 1) to simulate the impact of backdiffusion on Fa(NO) and to estimate the alveolar concentration actually due to in situ production (Fa(NO,prod)); and 2) to determine actual alveolar production in stable asthma patients with a broad range of NO bronchial productions. A model incorporating convection and diffusion transport and NO sources was used to simulate Fa(NO) and exhaled NO concentration at 50 ml/s expired flow (Fe(NO)) for a range of alveolar and bronchial NO productions. Fa(NO) and Fe(NO) were measured in 10 healthy subjects (8 men; age 38 +/- 14 yr) and in 21 asthma patients with stable asthma [16 men; age 33 +/- 13 yr; forced expiratory volume during 1 s (FEV(1)) = 98.0 +/- 11.9%predicted]. The Asthma Control Questionnaire (Juniper EF, Buist AS, Cox FM, Ferrie PJ, King DR. Chest 115: 1265-1270, 1999) assessed asthma control. Simulations predict that, because of backdiffusion, Fa(NO) and Fe(NO) are linearly related. Experimental results confirm this relationship. Fa(NO,prod) may be derived by Fa(NO,prod) = (Fa(NO) - 0.08.Fe(NO))/0.92 (Eq. 1). Based on Eq. 1, Fa(NO,prod) is similar in asthma patients and in healthy subjects. In conclusion, the backdiffusion mechanism is an important determinant of NO alveolar concentration. In stable and unobstructed asthma patients, even with increased bronchial NO production, alveolar production is normal when appropriately corrected for backdiffusion.     

Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and interferon-gamma

To test the hypothesis that tissue macrophages from AIDS patients have no intrinsic defects in either antimicrobial activity or in the capacity to respond to T cell-derived activating stimuli, alveolar macrophages from 11 patients were treated with crude lymphokines produced by healthy donors. After 72 hr of pretreatment with 10% mitogen- or antigen-induced crude lymphokines (which contained 300 U/ml of interferon-gamma [IFN-gamma]), AIDS alveolar macrophages generated twofold to threefold more H2O2 and readily inhibited the replication of the intracellular pathogens Toxoplasma gondii and Chlamydia psittaci. These responses were indistinguishable from those displayed by activated alveolar cells from 12 non-AIDS patients and three healthy volunteers. As judged by the abrogating effects of a neutralizing anti-human IFN-gamma monoclonal antibody, lymphokine-induced alveolar macrophage activation appeared to be largely IFN-gamma-dependent; thus, macrophages were also stimulated with recombinant (r)IFN-gamma alone. Seventy-two hours of treatment with 300 U/ml of rIFN-gamma resulted in both enhanced oxidative and antimicrobial activity comparable to that achieved by crude lymphokines, and the responsiveness of AIDS alveolar macrophages to rIFN-gamma was identical to control cells. These in vitro results suggest that tissue mononuclear phagocytes from AIDS patients a) are free of apparent defects in intracellular antimicrobial activity, b) are fully responsive to activating T cell products, and c) support the use of IFN-gamma as a potential macrophage-activating immunotherapeutic agent in AIDS-related opportunistic infections.     

Prolactin after baclofen in healthy subjects and prolactinoma patients

Serum prolactin (PRL) levels in basal conditions (two samples) and 30, 60, 90, 120, 150 e 180 minutes after oral administration of baclofen (20 mg) were evaluated in 6 healthy subjects and in 10 patients with prolactinoma. The effect of baclofen (20 mg by mouth) on the PRL secretion cimetidine (400 mg i.v.) or domperidone (20 mg i.v.) induced were evaluated in 9 healthy women by administration of baclofen 60 minutes before cimetidine or domperidone. Baclofen was unable to significantly rise serum PRL levels in healthy subjects and in patients affected by prolactinoma and furthermore did not interfere with PRL rise domperidone induced. On the contrary baclofen decreased PRL rise cimetidine induced. It was concluded that: in basal condition, GABAb receptor don't play an obvious role in modulation of PRL secretion; when H2 istaminergic inhibition on PRL secretion is blocked (at an hypothalamic site), a GABA inhibition, b receptor mediated, on PRL secretion became more clear; the domperidone blockade of hypophysial dopaminergic receptors suggests that GABAb modulation of prolactin secretion don't obtain itself by dopaminergic pathways.     

Arachidonic acid metabolism in alveolar macrophages. A comparison of cells from healthy subjects, allergic asthmatics, and chronic bronchitis patients

Arachidonic acid (AA) metabolism was studied in preparations of purified human alveolar macrophages (AM) from healthy subjects (HS = 5), allergic asthmatics (ABA = 9) and chronic bronchitis patients (CB = 7). AM incubated for 6 to 24 h in the presence of labeled AA and for an additional 5 h without labeled AA, released cyclooxygenase and lipoxygenase products into the medium. The study of the metabolites showed that the most abundant sulfidopeptide-leukotriene, was LTD4 as analyzed by TLC and identified by reversed phase HPLC. The release of LTD4 was time-dependent but it was shown to be significantly higher (p less than 0.01) in AM from ABA or CB than in those from HS. TLC analysis of radioactivity distributed between the different lipid classes at 24 h revealed more labeling in AM phospholipids from ABA and CB than in those from HS, and was reflected in phosphatidylethanolamine and phosphatidylinositol species. After 5 h without labeled AA the distribution was marked, by different in triglycerides, with a greater proportion of radioactivity in the control cells than in the pathological macrophages. Thus, the pathological lung state is an important factor affecting the release of LTD4 and the distribution of AA into cellular phospholipids. The differences observed between HS and ABA or CB phospholipid distribution suggests the existence of 2 different sources of AA release, one for inflammatory macrophages and another for quiescent cells.     

Aryl hydrocarbon hydroxylase activity in pulmonary alveolar macrophages and lymphocytes from lung cancer and noncancer patients: A correlation with family histories of cancer

Aryl hydrocarbon hydroxylase (AHH) activity was measured in pulmonary alveolar macrophages (PAMs) and peripheral blood lymphocytes from cigarette smokers with and without primary lung cancer. Frequency distribution analysis of AHH induction ratios for the two groups revealed an increased number of individuals in the lung cancer patient group with high lymphocyte induction values (P less than 0.05). A similar increase was not shown for high-PAM AHH values in lung cancer patients (P greater than 0.2). When individual PAM and lymphocyte AHH values were compared between noncancer and lung cancer patients, a positive correlation was observed for noncancer patients (r=0.195, P less than 0.001), but no correlation of these values was noted for lung cancer patients. The lung cancer patients were divided into three subgroups of patients showing (I) high PAM and low lymphocyte AHH levels, (II) low PAM and low lymphocyte AHH levels, and (III) low PAM and high lymphocyte AHH levels. When the incidence of family history of cancer was compared for these subgroups, no family cancer history was recorded for persons in subgroup II; however, individuals in subgroups I and III presented family cancer history incidence of 9.5% and 39.3%, respectively. Patients in group III averaged 6 years younger than those in group I. These data suggest that familial factors may be identified among lung cancer patients and that these factors appear to associate as either a cause of an effect with the capacity of pulmonary alveolar macrophages and lymphocytes to be induced for AHH. The data support the hypothesis that high AHH values may be characteristic of lung cancer patients but show that enzyme values determined from a single tissue, either PAMs or lymphocytes, may not be appropriate for showing whether high AHH inducibility is correlated with lung cancer.     

Metabolism of phosphatidylglycerol by alveolar macrophages in vitro

In whole animal studies, it has been shown that turnover of surfactant dipalmitoylphosphatidylglycerol (DPPG) is faster than that of dipalmitoylphosphatidylcholine (DPPC). The goal of this investigation was to characterize the metabolism of DPPG by alveolar macrophages and to determine whether they contribute to the faster alveolar clearance of DPPG. Isolated rat alveolar macrophages were incubated with liposomes colabeled with [(3)H]DPPG and [(14)C]DPPC. Macrophages internalized both lipids in a time- and temperature-dependent manner. The uptake of both lipids was increased by surfactant protein (SP) A and by adherence of the macrophages to plastic slides. The isotope ratio of DPPC to DPPG internalized by macrophages in suspension in the absence of SP-A was significantly lower than the isotope ratio in liposomes, suggesting that macrophages preferentially internalize DPPG when SP-A is absent. Phospholipase activity in macrophage homogenate was higher toward sn-2-labeled DPPG than toward sn-2-labeled DPPC. These studies show that alveolar macrophages play an important role in catabolizing surfactant lipids and may be partially responsible for the relatively faster clearance of DPPG from the lung.     

Hyaluronic acid-degrading enzymes in rat alveolar macrophages and in alveolar fluid: stimulation of enzyme activity after oral treatment with the immunomodulator RU 41740

RU 41740 (Biostim) is an immunomodulator clinically used for the treatment of chronic bronchitis and recurrent pulmonary infections. In these diseases large amounts of mucus are produced which congest the bronchi. A major glycosaminoglycan constituent of this mucus is hyaluronic acid, one of the largest molecules in nature; its metabolic degradation is carried out by 3 acid hydrolases: hyaluronidase, beta-N-acetylglucosaminidase, and beta-glucuronidase. In the lung these enzymes are especially synthesized and active in alveolar macrophages. It was thus interesting to study the effect of RU 41740 administration on the hyaluronic acid-degrading activity of these cells. This compound was given by gastric gavage to rats and the activities of lung alveolar macrophage and alveolar fluid hyaluronidase, beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase as a lysosomal marker were determined. The effect on macrophage proliferation was also examined. The results obtained showed that: (1) unstimulated alveolar macrophages display the remarkable property, compared with other cell types, that hyaluronidase activity is about equally distributed between the inside and the outside of the cell; (2) RU 41740 administration increases the total activity of the 4 enzymes studied in the alveolar macrophages without inducing any increase in the number of macrophages; (3) the intracellular activities of beta-N-acetylglucosaminidase and beta-glucuronidase are markedly increased, whereas intracellular hyaluronidase activity is not changed. However, in the extracellular fluid only hyaluronidase activity is highly increased; (4) even the lysosomal marker enzyme acid phosphatase has only its intracellular activity increased. This would suggest the possibility that other lysosomal enzymes may also be increased by this immunomodulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

ICAM-1-dependent fibroblast-lymphocyte adhesion: discordance between surface expression and function of ICAM-1

We have previously reported that stimulation of human fibroblasts (FB) with interferon-gamma (IFN-gamma) leads to their increased adhesiveness for resting peripheral blood T lymphocytes. With the use of blocking monoclonal antibodies, we determined that intercellular adhesion molecule-1 (ICAM-1) and its T cell ligand, lymphocyte function-associated antigen-1 (LFA-1) are the major, if not only ligands involved in this system. Using an ELISA, we have confirmed earlier reported observations that IFN-gamma induces an increase of ICAM-1 expression on the surface of FB suggesting that this increase mediates lymphocyte adhesion. However, we show that treatment of FB with IL-1, while leading to comparable increases in ICAM-1 synthesis and expression, failed to induce increased adhesion. In contrast, treatment of fibroblasts with the phorbol ester, TPA, stimulated ICAM-1-dependent adhesion without an increase in ICAM-1 surface expression. This suggested that the detection of ICAM-1 by monoclonal antibody techniques may not always correlate with its functional capabilities. The contrasting effects of IFN-gamma and IL-1 on ICAM-1-dependent FB adhesion suggest that qualitative as well as quantitative alterations of the ICAM-1 molecule may regulate ligand interaction.     

Shedding of soluble ICAM-1 into the alveolar space in murine models of acute lung injury

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in abundance on the cell surface of type I alveolar epithelial cells (AEC) in the normal lung and is a critical participant in pulmonary innate immunity. At many sites, ICAM-1 is shed from the cell surface as a soluble molecule (sICAM-1). Limited information is available regarding the presence, source, or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. We found sICAM-1 in the bronchoalveolar lavage (BAL) fluid of normal mice (386 +/- 50 ng/ml). Additionally, sICAM-1 was spontaneously released by murine AEC in primary culture as type II cells spread and assumed characteristics of type I cells. Shedding of sICAM-1 increased significantly at later points in culture (5-7 days) compared with earlier time points (3-5 days). In contrast, treatment of AEC with inflammatory cytokines had limited effect on sICAM-1 shedding. BAL sICAM-1 was evaluated in in vivo models of acute lung injury. In hyperoxic lung injury, a reversible process with a major component of leak across the alveolar wall, BAL fluid sICAM-1 only increased in parallel with increased alveolar protein. However, in lung injury due to FITC, there were increased levels of sICAM-1 in BAL that were independent of changes in BAL total protein concentration. We speculate that after lung injury, changes in sICAM-1 in BAL fluid are associated with progressive injury and may be a reflection of type I cell differentiation during reepithelialization of the injured lung.     

Tumor necrosis factor production in HIV-seropositive subjects. Relationship with lung opportunistic infections and HIV expression in alveolar macrophages

We have evaluated the TNF production by alveolar macrophages (AM) in 43 HIV-infected subjects in relation with 1) their clinical and biologic status; 2) the presence of lung opportunistic infections (OI); and 3) the expression of HIV by AM. This production was assessed in a standard chromium release test, using monocytic U937 cells as targets. The spontaneous TNF production by AM from patients without lung OI was higher than that from seronegative controls (p less than 0.02). This production by AM was similar to that of blood monocytes, suggesting that it was not related, in these subjects, to any particular lung status. The extent of TNF release by AM was correlated to the presence of a lymphocytic alveolitis (p less than 0.05), and not to the patients' clinical presentation nor to their CD4 cell count. Finally, AM from these subjects could be normally stimulated in vitro by IFN-gamma. On the other hand, it appeared that the spontaneous TNF release by AM shown in vitro to express HIV (p24+ AM) was significantly higher than that by their p24- counterparts (p less than 0.05) and by controls (p less than 0.01). In addition, contrasting with the marked increase of TNF release by p24- AM after their stimulation with IFN-gamma (p less than 0.001), p24+ AM appeared to be refractory to any stimulation by IFN, arguing for their activation in vivo. Finally, the spontaneous TNF release by AM was significantly increased during lung OI, compared with controls (p less than 0.01) as well as with AIDS patients without OI (p less than 0.01). In addition, the production of TNF by AM in these subjects was higher than that by the corresponding blood monocytes (p less than 0.02), suggesting a compartmentalization of this response within the lungs. In conclusion, it appears that the TNF production by AM of seropositive patients is highly related to the presence of lung OI as well as to the expression of HIV by these cells. In the context of the up-regulation of HIV expression induced by TNF in vitro, our data could suggest that the in vivo release of TNF by AM could participate in viral dissemination. Moreover, we hypothesize that the generation of activated AM refractory to any further stimulation could in turn lead to the development of additional pulmonary infections.     

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.     

Enhanced interleukin 1 production by alveolar macrophages and increase in Ia-positive lung cells in silica-exposed rats

Inhalation exposure to silica dust enhanced interleukin 1 (IL-1) production by alveolar macrophages (AM), which is attributable to an increase in Ia-positive lung cells. While the proportion of Ia-positive cells in lavaged bronchoalveolar cells (BAC) was much lower (0-3%) in unexposed control rats, about a third of the rats that inhaled silica showed higher proportions (8.0-18.5%); these were designated "Ia-high" exposed animals. The number of total cells, Ia-positive cells and lymphocytes in BAC was significantly increased (P less than 0.05, P less than 0.001, and P less than 0.001, respectively) in these "Ia-high" exposed animals, compared to the control animals. Adherent AM populations obtained from BAC preparations also contained significantly higher (P less than 0.001) proportions of Ia-positive cells in the "Ia-high" exposed animals. When these adherent AM cultures were stimulated with lipopolysaccharide, IL-1 activity of the culture supernatants was enhanced and was significantly higher (P less than 0.001) in the "Ia-high" exposed rats, compared to the control animals. These results indicate that silica-exposure can induce populational changes in lung cells and also activation of AM associated with the increase in Ia-positive cells.     

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Superoxide production from macrophages of leprosy patients after stimulation withMycobacterium leprae

The macrophages from peripheral blood of normal healthy individuals respond to live or killedMycobacterium leprae by producing superoxide. On the other hand, the macrophages from bacteriologically positive (B +LL) or long term treated bacteriologically negative (B -LL) and tuberculoid leprosy patients are unable to produce superoxide when stimulated with liveMycobacterium leprae. While killedMycobacterium leprae induce superoxide with the cells from tuberculoid andB(-)LL patients, cells fromB(+)LL patients fail to respond. The deficiency inB(-)LL patients to produce superoxide appears to be specific withMycobacterium leprae and the defect can be counteracted by the addition of colchicine. These observations indicate a preexisting membrane disposition which does not favour superoxide production. A similar situation is seen in the cells from tuberculoid leprosy patients. Thus it appears that both cured and active lepromatous leprosy patients have defective macrophages, unable to respond to liveMycobacterium leprae to produce superoxide anion, in contrast to the situation with the cells from normal healthy individuals.