Normal Alveolar Macrophages Research Articles

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis.

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.

Oxidative Metabolism in the Alveolar Macrophage: Analysis by Flow Cytometry

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non-fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium. Alveolar macrophages (AMs) loaded with either DCFH or HE were analyzed after phorbol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer, show that both DCFH (273 +/- 48, mean increase over control +/- SE, fmol/cell, N = 9) and HE (416 +/- 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AMs ingesting opsonized fluorescent latex beads was measured in subpopulations ingesting increasing numbers of particles. A graded increase in oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylyococcal bacteria caused similar changes in HE-loaded AMs. Inhibition of respiration with antimycin showed that approximately 95% of the increased oxidative metabolism of hamster AMs ingesting opsonized beads or bacteria was mitochondrial. The remaining 5% (10-40 fmol/cell) is membrane-derived oxidative activity quantitatively similar to that measured in assays of extracellular release of H2O2. Monocytes loaded with either DCFH or HE showed substantial increases in fluorescence after PMA stimulation (mean % increase over control +/- SE at 30 min: 464 +/- 104, DCFH, 505 +/- 156, HE). While DCHF is known to measure H2O2, HE is less well characterized. Exposure of cells to an extracellular source of both superoxide anion (O2-) and H2O2, xanthine oxidase-xanthine, resulted in marked oxidation of intracellular HE. Addition of both superoxide dismutase and catalase blocked this oxidation, indicating that HE can detect both O2- and H2O2. These agents can be useful probes for precise analysis of oxidative metabolism during phagocytosis in AMs and other mononuclear phagocytes.

Tumor necrosis factor‐alpha induces vitamin D‐1‐hydroxylase activity in normal human alveolar macrophages

The induction of 1-hydroxylase in alveolar macrophages by tumor necrosis factor-alpha (TNF) was examined in view of recent evidence suggesting that local production of 1,25-(OH)2D3 may play a role in the regulation of immune functions. Incubation of pulmonary alveolar macrophages from normal human subjects with recombinant TNF caused a 2- to 10-fold increase in 25-hydroxyvitamin D3-1-hydroxylase activity. The dose-response curve was linear over the range 0.05-5.0 IU/ml, and no further increase was seen at higher concentrations. The increase in 1-hydroxylase activity was present after 12 h and reached a maximum after 3 days. The effect of TNF was inhibited in a dose-dependent manner by the presence of 1,25(OH)2D3 (10(-10)-10(-8) M) in the incubation media for 5 days but was unaffected by 10(-9) M 1,25(OH)2D3 after 12 h. The enhancement of macrophage 1-hydroxylase activity by TNF was comparable to that induced by gamma interferon (IFN) but the effects of maximal doses of both agents were not additive. The presence of antibody to TNF resulted in a 76% inhibition of TNF-induced 1-hydroxylase but had no significant effect on IFN-induced 1-hydroxylase activity.

Arachidonic Acid Metabolism in Normal Human Alveolar Macrophages: Stimulus Specificity for Mediator Release and Phospholipid Metabolism, and Pharmacologic Modulation In Vitro and In Vivo

Arachidonic acid (AA) metabolism in normal human alveolar macrophages including phospholipid turnover, stimulus specificity for mediator release including trigger synergy, and the pharmacologic control of the release of AA metabolites was explored. Macrophages labeled overnight with [3H]AA, then activated, released three major AA metabolites, thromboxane B2 (TxB2), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE), as characterized by thin layer chromatography, high performance liquid chromatography, and radioimmunoassay. Although all triggers (phorbol myristate acetate [PMA], serum-activated zymosan, and ionophore A23187) resulted in the release of TxB2 and free AA, efficient synthesis of lipoxygenase products, particularly LTB4, required A23187. A23187 was the most effective single agent in producing LTB4 synthesis, was synergistic with PMA in causing LTB4 release, and was associated with significant turnover of phosphatidylcholine and phosphatidylinositol. Incubation of macrophages in vitro with cyclooxygenase inhibitors resulted in an inhibition of the formation of cyclooxygenase products; however, no shunting of metabolites into products of the lipoxygenase pathway was observed. Although overnight incubation of macrophages in vitro with dexamethasone (1 microM) resulted in an inhibition of both the spontaneous and A23187/PMA-triggered release of all AA metabolites, treatment of 5 volunteers with dexamethasone (4 mg po bid x 7 doses, in a single-blind, placebo-controlled, crossover protocol) resulted in no significant inhibition of the release of AA metabolites from macrophages triggered ex vivo. We conclude that activation of normal human alveolar macrophages results in phospholipid turnover (phosphatidylcholine and phosphatidylinositol) and the release of three major AA metabolites (TxB2, LTB4, and 5-HETE); that optimal synthesis of lipoxygenase product requires the presence of a calcium signal (A23187), although PMA can synergize with A23187 in the production of lipoxygenase products; and that glucocorticoids may have a different effect on the release of AA metabolites from alveolar macrophages when administered in vitro versus in vivo.

Normal Human Alveolar Macrophages Have More Ability than Blood Monocytes to Produce Cell-associated Interleukin-1-alpha

Human alveolar macrophages (AM) were obtained by bronchoalveolar lavage from healthy donors, and their abilities to produce extracellular and cell-associated interleukin 1 (IL-1) in response to various activation stimuli were compared with those of autologous blood monocytes. The production of IL-1 alpha and IL-1 beta by monocytes and AM was examined by thymocyte co-stimulation assay and enzyme immunoassays (EIA). Results showed that when activated with lipopolysaccharide (LPS) or desmethyl muramyl dipeptide (norMDP), AM released much less extracellular IL-1 beta than did blood monocytes. In contrast, these activated AM produced more cell-associated IL-1 than did blood monocytes. When the IL-1 activity was examined by the thymocyte assay, the extracellular and cell-associated IL-1 produced by the two cell types were largely IL-1 beta and IL-1 alpha, respectively, as shown by antibody neutralization. The cell-associated IL-1 activity of AM induced by the synergistic actions of suboptimal concentrations of recombinant interferon-gamma (rIFN-gamma) and norMDP was also higher than that of autologous blood monocytes. Consistent with these findings on AM, macrophages generated in vitro by maturation of blood monocytes produced higher levels of cell-associated IL-1 activity than did freshly isolated monocytes. These observations suggest that AM may play a critical role in situ regulation of pulmonary inflammatory and immune reactions through production of cell-associated IL-1 alpha.

Enhanced Accessory Cell Function by Alveolar Macrophages from Patients Infected with the Human Immunodeficiency Virus: Potential Role for Depletion of CD4+ Cells in the Lung

Mononuclear phagocytes, including alveolar macrophages (AM), can be infected by the human immunodeficiency virus (HIV). Acting as accessory cells (AC), AM could infect CD4 lymphocytes through cell-to-cell contact and by inducing T cell proliferation, which increases lymphocyte susceptibility to infection. Using normal allogeneic T cells as responders, AM from infected individuals demonstrated an enhanced ability to stimulate a Con A and pokeweed mitogen lymphocyte proliferation assay compared with normal AM. Exogenous IL 1 enhanced the stimulation of a mitogen response by normal AM, but not from HIV-positive individuals, suggesting increased levels of this cytokine may explain the observed enhancement. However, increased IL 1 secretion by AM from HIV-infected patients could not be demonstrated, either in a bioassay or antigenically using an ELISA for IL-1 beta. Syncytia formation was observed when AM from asymptomatic HIV-positive individuals were cultured with normal T cells, suggesting viral transmission was occurring. Finally, in individual patients the stimulation of a mitogen response was inversely correlated with the CD4/CD8 ratio and total CD4 count, suggesting that enhanced AC function and CD4 cell depletion may be related in vivo. These findings indicate that enhanced AM accessory cell function is seen in HIV-infected individuals and could be a potential mechanism for CD4 cell depletion in the lung.

SV40-transformed Human Lung Fibroblasts Secrete a 92-kDa Type IV Collagenase Which Is Identical to That Secreted by Normal Human Macrophages

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.

Pulmonary alveolar macrophages from patients with active sarcoidosis express type IV collagenolytic proteinase. An enzymatic mechanism for influx of mononuclear phagocytes at sites of disease activity.

Alveolar macrophages (AMs) recovered from the bronchoalveolar lavage (BAL) of 44 patients with sarcoidosis were evaluated for their ability to release type IV collagenolytic metalloproteinase (IV-Case). This enzyme, which is produced by peripheral blood monocytes (PBMs) but not by tissue macrophages, degrades type IV collagen, the major structural component of vessel wall basement membranes, and helps to promote the migration of PBMs from the blood compartment to peripheral tissues. Our results demonstrated that AMs from patients with active sarcoidosis released significantly increased levels of IV-Case with respect to patients with inactive disease and control subjects. After in vitro culture, sarcoid AMs secreted IV-Case during the first 24 h of collection; after that time, AMs progressively lost their ability to release IV-Case. Exposition of both sarcoid and normal AMs to recombinant IL 2 or gamma IFN did not influence their property to release IV-Case. The immunoblot analysis of IV-Case demonstrated complete identity between IV-Case released by AMs and the degradative enzyme obtained from PBMs. The increased property to release IV-Case was significantly related to the increase of the absolute number of AMs and, in particular, of AMs bearing two determinants that are usually expressed by most PBMs (CD11b and CD14). Selective depletion of CD11b+/CD14+ AMs from the entire macrophagic population was associated with the recovery of the IV-Case activity to normal values. A positive correlation was also found between the increase in the absolute number of lung T cells and the enhanced CD4/CD8 pulmonary ratio. A 6-mo follow-up study indicated a significant association between the positivity for the 67Gallium scan and the increased property of AMs to release IV-Case. Our data are consistent with the hypothesis that a IV-Case mediated influx of peripheral monocytes takes place in the lung of sarcoid patients. Furthermore, the correlation found between the IV-Case release and disease activity suggests that this assay could represent a useful tool in sarcoidosis disease staging.

Expression of HLA Class II Genes in Alveolar Macrophages of Patients with Sarcoidosis

Sarcoidosis is characterized by the accumulation of activated helper/inducer T-cells and macrophages at sites of disease. The accumulation of these T-cells may be related to the ability of sarcoid alveolar macrophages to present antigen and stimulate lymphocyte proliferation in an exaggerated fashion. In the context that HLA Class II gene (HLA-DR, DQ, and DP) expression is essential to the interaction of lymphocytes with antigen-presenting cells such as macrophages, we have evaluated the hypothesis that the expression of Class II genes may be up-regulated in sarcoid alveolar macrophages. Northern blot and dot blot analysis with 32P-labeled cDNA probes for HLA-DR, DQ, and DP genes revealed that both normal and sarcoid alveolar macrophages contain Class II mRNA transcripts, but that the levels in sarcoid and normal alveolar macrophages are similar (p greater than 0.3, all comparisons). Furthermore, evaluation of surface expression of Class II molecules with monoclonal antibodies and flow cytometry analysis demonstrated that alveolar macrophages of both patients with sarcoidosis and normal subjects express all three Class II molecules, DR, DQ, and DP, but there were no significant differences between sarcoid patients and normal subjects in the proportions of macrophages expressing these surface molecules. However, there was a significantly higher surface density of Class II molecules on sarcoid alveolar macrophages (p less than 0.05, all comparisons sarcoid to normal).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide stimulates alveolar macrophage adherence in vivo and in vitro.

The number of alveolar macrophages (AM) obtained from rats by bronchoalveolar lavage (BAL) decreases significantly within two hours after infusion of E. coli lipopolysaccharide (LPS). To investigate the mechanism of this phenomenon, we evaluated adherence properties of AM obtained from rats 4 hours after intravenous injection of E. coli LPS (3 mg/kg) or normal saline (NS). Significantly fewer AM were present in BAL samples from LPS-treated rats (4.09 x 10(6] compared to NS-treated rats (7.55 x 10(6]. When a 17 mM lidocaine solution (LDC) was used for lavage, the number of AM in BAL samples from LPS-treated rats increased significantly (7.38 x 10(6]. Adherence of AM from LPS-treated rats to rotating plastic tubes was significantly greater than that of AM from NS-treated rats. Incubation of normal AM with LPS (5 or 15 micrograms/ml) in serum-free media increased AM adherence to plastic. LPS-induced adherence was significantly greater at the higher LPS dose. Thus, the decreased number of AM obtained by BAL of LPS-treated rats appears to be due to increased adherence of AM to alveolar structures. This phenomenon may be mediated by direct effects of LPS on AM.

Unilateral radiation pneumonitis in sheep: physiological changes and bronchoalveolar lavage

Radiation pneumonitis is a life-threatening result of therapeutic thoracic irradiation, yet its mechanisms are poorly understood. We studied the effects of unilateral lung irradiation (3,000 rad) in sheep from the immediate response to the later development of radiation pneumonitis. We defined radiation pneumonitis by its diagnostic clinical feature, radiographic infiltration of the irradiated zone with a straight margin corresponding to the radiation port. The immediate response in the few hours after irradiation was characterized by cough, labored respiration, hypoxemia (arterial PO2 decreased 19 Torr), mild pulmonary hypertension (pulmonary arterial pressure increased 20%), and lymphopenia. Hemodynamics and gas exchange returned to normal by day 2 but became abnormal again before or during radiation pneumonitis at 32 +/- 2 days. Respiratory distress, hypoxemia, and pulmonary hypertension recurred during radiation pneumonitis. Bronchoalveolar lavage during radiation pneumonitis contained increased neutrophils (19 +/- 4%, control = 7%), increased protein (0.27 +/- 0.1 g/dl, control = 0.12 +/- 0.03), and severely impaired ability to lower surface tension. Alveolar macrophages from both lungs during unilateral radiation pneumonitis exhibited impaired generation of superoxide after phorbol myristate (only a 30% increase). Normal control alveolar macrophages increased superoxide production after stimulation greater than 400%. We conclude that unilateral lung irradiation in sheep causes a mild immediate response followed by radiation pneumonitis at 1 mo. Unilateral radiation pneumonitis in this model is associated with ipsilateral neutrophilic alveolitis, increased bronchoalveolar lavage protein, and impaired surfactant function, as well as bilateral functional abnormalities of alveolar macrophages.

Effects of Extracellular Slime Produced by Staphylococcus epidermidis on Oxidative Responses of Rabbit Alveolar Macrophages

Bacterial slime produced in mass cultures of the RP 12 strain of Staphylococcus epidermidis was extracted with 4 M guanidine-HCl plus 0.05 M sodium acetate and 0.5% CHAPS, concentrated, dialyzed, and subjected to separation on DEAE sephacel columns. Three fractions, I-2A, I-2B, and I-4, were eluted with linear gradients of NaCl. Fractions I-2A and I-2B were alcian blue positive, whereas I-4 was alcian blue negative but the most electronegative fraction. The crude polysaccharide fraction and the three purified fractions were incubated individually for 2.5 or 20 h with normal rabbit alveolar macrophages (AM) to determine their effect on a subsequent PMA-induced oxidative burst. The crude fraction (50-200 micrograms/mL) and I-2B (50-200 micrograms/mL) primed the AM to give approximately a threefold increase in the PMA-induced burst after 2.5 h incubation. In contrast, a 20-h incubation resulted in a 30-40% inhibition of the PMA-induced burst with AM incubated with the same concentrations of the crude, I-2A, or I-2B fractions. Fraction I-4 had no detectable effect. The fractions also were tested to determine if they could elicit an oxidative burst in BCG-immune AM. None of the fractions (up to 500 micrograms/mL) elicited a significant oxidative burst even though BCG-immune AM yielded a PMA-induced burst 100 times that observed with normal resident AM. These data suggest that slime from S. epidermidis can impair the PMA-induced oxidative burst of normal AM during a 20-h incubation period and could explain in part why host defenses are compromised by slime-producing S. epidermidis.

Evidence for the presence of angiotensins in normal, unstimulated alveolar macrophages and monocytes.

The presence of angiotensins was demonstrated in normal unstimulated alveolar macrophages and monocytes from both mice and rats. These peptides were partially purified from cell homogenates by ion exchange chromatography and identified as being [Ile5] angiotensin I (Ang I), [Ile5] angiotensin II (Ang II) and to a lesser extent [Ile4] angiotensin III (Ang III) using high performance liquid chromatography (HPLC). Based on the present data both alveolar macrophages and monocytes expressed Ang I as quantified by a specific and sensitive radio-immunoassay (RIA) from the HPLC eluates. In contrast to this, alveolar macrophages from both mice and rats exhibited a fairly low, if detectable Ang II content. It seems reasonable to suggest that, in contrast to monocytes, macrophages do not generate and/or incorporate Ang II appreciably, at least in their resting stage. Although it is still not obvious whether these mononuclear phagocytes generate or simply capture angiotensin(s) from the blood pool or from the tissues; they must serve as in vivo target cells for the angiotensin system, at least for the plasma or tissue clearance of these molecules.

Bronchoalveolar Macrophages from Patients with Idiopathic Pulmonary Fibrosis Are Unable To Kill Facultative Intracellular Bacteria

The accumulation of inflammatory and immune effector cells in the lungs of patients at early stages of interstitial lung disease has been well documented, but little is known about the functional activity of these cells, particularly alveolar macrophages. The purpose of the experiments described here was to determine whether alveolar macrophages from patients with idiopathic pulmonary fibrosis (IPF) differed from alveolar macrophages of normal subjects using a model system that assesses a component of cell-mediated immunity. Alveolar macrophages were tested for their ability to phagocytose and kill the facultative intracellular bacterium Listeria monocytogenes. Because this system requires the stimulation of macrophages by antigen-specific T-cells, it allows the assessment of effector functions of macrophages found in the lower respiratory tract of patients with IPF. Data showed that alveolar macrophages from normal subjects both phagocytosed and killed those bacteria. However, alveolar macrophages from patients with IPF phagocytosed bacteria normally but expressed little bactericidal or bacteriostatic activity. Further, we found no difference in HLA-DR expression by normal and IPF alveolar macrophages. Therefore, it does not appear that this defect in bactericidal activity in IPF macrophages results from a defect in the antigen-presenting function of these cells. These data suggest that alveolar macrophages from patients with IPF express a functional deficiency in their ability to kill facultative intracellular bacteria.

Tissue-Specific Expression and Developmental Regulation of the Human fgr Proto-Oncogene

In this study, we show that c-fgr proto-oncogene expression is limited to normal peripheral blood granulocytes, monocytes, and alveolar macrophages, all of which contain 50 to 100 copies of c-fgr mRNA per cell. The c-fgr RNA molecules in these cells consisted of partially spliced transcripts containing intron 7 and completely spliced molecules capable of encoding the predicted p55 c-fgr protein. The splicing of intron 7 appeared to occur after the splicing of most of the other introns; partially spliced molecules containing intron 7 did not appear to be transported into the cytoplasm. Very low levels of fgr transcripts were also present in U937 promonocytic cells and increased in abundance with 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. The level of fgr transcripts began to increase 2 to 4 h after TPA addition, peaked at 8 h, and subsequently declined. Since we found that the half-life of fgr mRNA was longer than 8 h, these changes are best explained by transient transcriptional activation of fgr during TPA-induced differentiation, although nuclear runoff experiments were not sensitive enough to detect this event. Cycloheximide also caused accumulation of c-fgr transcripts in U937 cells; no superinduction was observed when TPA and cycloheximide were added at the same time. Induction by either agent was blocked with actinomycin D. These results demonstrate that the c-fgr gene is expressed in a tissue- and development-specific fashion and suggest that constitutive expression of c-fgr in U937 cells is regulated by a labile transcriptional repressor.

Tissue-specific expression and developmental regulation of the human fgr proto-oncogene.

In this study, we show that c-fgr proto-oncogene expression is limited to normal peripheral blood granulocytes, monocytes, and alveolar macrophages, all of which contain 50 to 100 copies of c-fgr mRNA per cell. The c-fgr RNA molecules in these cells consisted of partially spliced transcripts containing intron 7 and completely spliced molecules capable of encoding the predicted p55 c-fgr protein. The splicing of intron 7 appeared to occur after the splicing of most of the other introns; partially spliced molecules containing intron 7 did not appear to be transported into the cytoplasm. Very low levels of fgr transcripts were also present in U937 promonocytic cells and increased in abundance with 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. The level of fgr transcripts began to increase 2 to 4 h after TPA addition, peaked at 8 h, and subsequently declined. Since we found that the half-life of fgr mRNA was longer than 8 h, these changes are best explained by transient transcriptional activation of fgr during TPA-induced differentiation, although nuclear runoff experiments were not sensitive enough to detect this event. Cycloheximide also caused accumulation of c-fgr transcripts in U937 cells; no superinduction was observed when TPA and cycloheximide were added at the same time. Induction by either agent was blocked with actinomycin D. These results demonstrate that the c-fgr gene is expressed in a tissue- and development-specific fashion and suggest that constitutive expression of c-fgr in U937 cells is regulated by a labile transcriptional repressor.

Use of Allophycocyanin Allows Quantitative Description by Flow Cytometry of Alveolar Macrophage Surface Antigens Present in Low Numbers of Cells

Autofluorescence has limited quantitative description by flow cytometry of certain alveolar macrophage surface antigens. Autofluorescence is most significant when macrophages are excited by blue light and emission is monitored in the green-yellow light range. Because of recent advances in flow cytometry and in the development of fluorescent labeling compounds, such as allophycocyanin, autofluorescence can be minimized by the use of red excitation and emission wavelengths. We have developed a three-step labeling technique that discretely identifies the minor subpopulation of normal rat alveolar macrophages that express Class II major histocompatibility (Ia) antigens. Using this technique, we observed that alveolar macrophage Ia antigen expression increases at least fourfold after intravenous administration of bacillus Calmette-Guerin to previously primed rats. Additionally, we observed that in vitro treatment of alveolar macrophages by exposure to gamma interferon results in an increase in Ia antigen expression. This new method is significant because flow cytometry is a powerful tool with which to analyze quickly, accurately, and reproducibly alveolar macrophage surface antigens.

Effects of in vitro exposure to nitrogen dioxide on human alveolar macrophage release of neutrophil chemotactic factor and interleukin-1

To evaluate the potential toxic and immunologic effects of nitrogen dioxide (NO2) exposure on cells from the lower respiratory tract, normal human alveolar macrophages obtained by bronchoalveolar lavage were exposed to increasing concentrations of NO2 using an in vitro exposure system. Alveolar macrophages exposed to 5, 10, or 15 ppm NO2 for 3 hr showed no difference in cell viability when compared to air-exposed macrophages. In addition, the spontaneous release of neutrophil chemotactic factor (NCF) was not changed by NO2 exposure, nor was there any effect on the ability of alveolar macrophages to release increased amounts of NCF following stimulation with activated zymosan. Furthermore, alveolar macrophages did not spontaneously release interleukin-1 (IL-1) following air or NO2 exposure. When stimulated with influenza virus both air- and NO2-exposed cells released increased amount of IL-1, but was no significant difference in the amount of IL-1 released by air- and NO2-exposed alveolar macrophages. Thus, although NO2 exposure is known to incite an inflammatory response in the lower respiratory tract, using the in vitro exposure system described in this study we were unable to demonstrate a direct toxic effect of NO2 on viability or any NO2-induced change in the release of the immunoregulatory molecules NCF and IL-1.

Production of plasminogen activator by alveolar macrophages in normal subjects and patients with interstitial lung disease.

Increased production of the serum protease plasminogen activator is associated with tissue damage. The in vitro production of plasminogen activator by alveolar macrophages obtained by bronchoalveolar lavage was studied in 22 normal subjects and 28 patients with interstitial lung disease to determine whether plasminogen activator is produced by normal alveolar macrophages and whether this is increased in patients with interstitial lung disease. Plasminogen activator activity, measured with an iodine-125 labelled fibrin release assay, was found to be dependent on time, effector cell numbers, and plasminogen concentration. Plasminogen activator production by alveolar macrophages from 14 normal non-smokers and eight normal smokers was similar and the mean value was 0.78 (SEM 0.16) urokinase (UK) units x 10(-8)/cell/hour. Alveolar macrophages from the seven patients with cryptogenic fibrosing alveolitis and six patients with histiocytosis-X produced more plasminogen activator (1.89 (0.25) and 4.54 (1.3) x 10(-8) UK units/cell/hour respectively) than macrophages from normal subjects (p less than 0.05), whereas those from 15 patients with sarcoidosis did not (1.09 (0.2) x 10(-8) UK units/cell/hour). Exposure of normal alveolar macrophages to immune complexes enhanced plasminogen activator production to 2.07 (0.27) x 10(-8) UK units/cell/hour, whereas exposure to products of activated T cells and to purified gamma interferon reduced plasminogen activator production (to 0.38 (0.11) and 0.62 (0.11) x 10(-8) UK units/cell/hour respectively). These studies show that plasminogen activator is produced by normal human alveolar macrophages and that its production is increased in patients with cryptogenic fibrosing alveolitis and histiocytosis-X.

Demonstration by in situ hybridization of dissimilar IL-1 beta gene expression in human alveolar macrophages and blood monocytes in response to lipopolysaccharide.

IL-1 beta is the major form of IL-1 produced by mononuclear phagocytes. To evaluate the possible mechanisms underlying the observation that mature populations of human mononuclear phagocytes as alveolar macrophages are relatively poor IL-1 producers compared with blood monocytes, the expression of the IL-1 beta gene mRNA transcripts was evaluated in LPS-stimulated normal autologous blood monocytes and alveolar macrophages by using a IL-1 beta cDNA probe. Although Northern analysis demonstrated that stimulated monocytes and alveolar macrophages both express 1.8-kb IL-1 beta mRNA transcripts, cytoplasmic dot blot analysis showed that the total IL-1 beta mRNA content in alveolar macrophages was only 38 +/- 5% of that in blood monocytes. In situ hybridization with antisense and sense IL-1 beta RNA probes demonstrated that whereas most of stimulated blood monocytes contained IL-1 beta mRNA transcripts, a significant proportion of autologous alveolar macrophages stimulated in an identical fashion did not express the IL-1 beta gene. Within 4 h after LPS stimulation, IL-1 beta mRNA transcripts were detected in 81 +/- 6% monocytes, whereas only 16 +/- 9% of alveolar macrophages were positive, and by 18 h this had increased only to 43 +/- 15%. Quantification of the size distribution of the IL-1 beta mRNA expressing mononuclear phagocytes demonstrated that, among the population of alveolar macrophages, the cells expressing this gene were not confined to those that were "monocyte-like." These observations demonstrate that there is a heterogeneity among population of mononuclear phagocytes in their ability to express the gene for IL-1 beta, which could explain the differences observed in IL-1 production.