Peroxidase activity of alveolar macrophages.

Peroxidase activity was studied in alveolar macrophages and compared to the peroxidase activity in polymorphonuclear leukocytes using cytochemical techniques. A dense reaction product for peroxidase was observed in the primary lysosomes of polymorphonuclear leukocytes, but no significant peroxidase or peroxidative enzymes could be detected in rabbit alveolar macrophages. Furthermore, following vigorous phagocytosis of zymosan particles by alveolar macrophages in vitro, no peroxidase could be detected in association with the phagocytic vacuole. Exogenous horseradish peroxidase was ingested readily by alveolar macrophages so that abundant reaction product was demonstrated in pinocytotic vesicles and phagocytic vacuoles. The uptake of exogenous peroxidase by pinocytosis appeared to be more vigorous in alveolar macrophages than in polymorphonuclear leukocytes. These studies demonstrate that alveolar macrophages do not contain significant quantities of peroxidase and suggest that, it contrast to polymorphonuclear leukocytes, peroxidative metabolism does not contribute in a major way to microbial killing by alveolar macrophages.     

Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease.

The effects of the 56-kilodalton protease (56K protease) from Serratia marcescens on complement-derived chemotactic activity were examined. Fresh human serum was incubated with zymosan to produce C5a. This activated serum was then incubated with various concentrations of 56K protease, and the chemotactic activity of mouse peritoneal exudate polymorphonuclear leukocytes (PMN) and macrophages was evaluated. A significant dose-dependent decrease of chemotactic activity was observed after protease treatment. Furthermore, treatment of human recombinant C5a with 56K protease at a dose of 1.0 microgram/ml resulted in a complete loss of chemotactic activity. When the living bacteria of the virulent strain, which produced about 10 times more protease than did the less virulent strain, were injected intraperitoneally into mice, the magnitude of infiltration of polymorphonuclear leukocytes into the peritoneal cavity was much lower than that caused by the less virulent strain. Because complement-dependent chemotactic activity is an initial response to bacterial infection, these results suggest indirect pathogenic functions of serratial proteases that suppress chemotactic activity.     

Lipid chemotaxins isolated from culture filtrates ofEscherichia coli and from oxidized lipids

Lipid extracts of sterile culture filtrates ofEscherichia coli were shown to contain approximately 75% of the chemotactic activity for human polymorphonuclear leukocytes and rabbit alveolar macrophages. Fractionation and purification of these lipids revealed the presence of many unknown lipids of widely different properties, but all were anionic and at very low concentrations, chemotactic. The only one of active molecules that could be identified was an unsaturated ultraviolet-absorbing hydroxy fatty acid, which, following catalytic reduction with hydrogen, was found to be hydroxyeicosanoic acid. This fatty acid's Chromatographic behavior was very similar to that of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which is a potent chemotaxin for polymorphonuclear leukocytes and macrophages. Unknown chemotaxins could be generated by the oxidation of known unsaturated lipids. Prostaglandins A₂ and E₂ produced potent chemotaxins upon aerobic oxidation. Malonaldehyde, a peroxidation product of unsaturated lipids, when reacted with phosphatidylethanolamine in aerobic conditions, also produced strong chemotactic agents. The chemotactic activity of these products could be destroyed by catalytic reduction with hydrogen and by methylation with dry methanolic HCl. These data indicate that the nonenzymatic oxidation of unsaturated lipids generates some products that are potent chemotaxins for mammalian inflammatory cells.     

New aspects of chemotaxis. Specific target-cell attraction by lipid and lipoprotein fractions of Escherichia coli chemotactic factor.

A chemotactic factor extracted from sterile filtrates of Escherichia coli cultures was strongly chemotactic for polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (RAM). Electrophoresis of the cytotactic material yielded five lipid fractions: one that was protein-free and active toward both PMN and RAM, and four lipid-protein complexes that were strongly chemotactic only for RAM. Thin-layer chromatography of the lipid-protein complexes resulted in an unmasking of PMN activity in a peptide-free lipid extract, while the isolated peptidic components were essentially noncytotactic. The original RAM activity was retained in the unmasked lipid, which possessed chemical and chromatographic properties similar to those of a previously reported cytotaxin synthesized from arachidonic acid. These data indicate that a class of lipids derived from bacterial and cellular sources is intrinsically cytotactic for PMN and RAM. When peptide moieties are associated with cytotactic lipids, the resultant lipid-peptide complex may exhibit cellular specificity not evident in the free lipid.     

Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis

Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection.     

Rat alveolar macrophage production of chemoattractants for neutrophils: response to Escherichia coli endotoxin.

Endotoxemia in rats is associated with the accumulation of neutrophils (polymorphonuclear leukocytes) within the airspaces of the lung. Polymorphonuclear leukocyte influx appears to be regulated by the intrapulmonary accumulation of chemotactic activity. Since alveolar macrophages (AMS) are prevalent cells in the airspace and are known to release a variety of chemotactic factors, we investigated the effect of endotoxin exposure on AM production of chemotactic activity. We tested the hypothesis that endotoxin-exposed AMs have an augmented ability to produce chemoattractants. We recovered AMs by bronchoalveolar lavage from control rats and from rats treated in vivo with a "low dose" (2.5 mg/kg) or a "high dose" (5.0 mg/kg) of Escherichia coli endotoxin. These AMs were then cultured in vitro for 15 h in the absence or the presence of endotoxin (15 and 30 micrograms/ml) to stimulate the cells to produce chemoattractants. We found that in vitro endotoxin stimulated normal AMs to secrete chemoattractants in a dose-dependent fashion. AMs from rats treated with endotoxin in vivo spontaneously secreted more chemoattractants than AMs from control rats. Exposure to in vivo endotoxin followed by in vitro stimulation with endotoxin resulted in an even greater production of chemoattractants by AMs. We found a significant association between the percent polymorphonuclear leukocytes recovered by bronchoalveolar lavage from the airspaces and the production of chemoattractants by AMs from the same specimen. The level of chemotactic activity spontaneously produced by AMs predicted the degree of stimulated production of chemotactic activity. Partial purification indicated that this chemotactic activity has two molecular weight peaks, one near 1,000 and the other near 50,000. The activity was stable at 100 degrees C for at least 30 min and was degradable by trypsinization. We conclude that endotoxin can induce AM production of chemoattractants and that prior exposure to endotoxin in vivo affects the response of AM to in vitro endotoxin exposure. By inference, it is possible that this endotoxin-macrophage interaction may serve as a biologic amplifier of the effects of endotoxin and may have a role in the pathogenesis of septic lung injury in humans.     

Interaction of Mycoplasma dispar and Mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leukocytes.

Mycoplasma dispar and Mycoplasma agalactiae subsp. bovis survived or grew in cultures of bovine lacteal polymorphonuclear leukocytes or bovine alveolar macrophages. In the presence of specific bovine antibody, macrophages and polymorphonuclear leukocytes appeared to kill both species of mycoplasma. Specific rabbit antisera also promoted the killing of these mycoplasmas by bovine macrophages but had no demonstrable activity for bovine polymorphonuclear leukocytes. It is suggested that phagocytosis of these mycoplasmas by bovine cells occurs only in the presence of specific antibody. The experiments also indicate that differences exist between bovine polymorphonuclear leukocytes and macrophages with regard to their receptor sites for immunoglobulins.     

Effect of phorbol myristate acetate on cellular metabolism and lysozyme release from alveolar macrophages and polymorphonuclear leukocytes

Phorbol myristate acetate stimulated oxidative metabolism in alveolar macrophages and blood neutrophils. This compound also stimulated lysozyme release from neutrophils but not from alveolar macrophages. These findings suggest that the regulation of lysozyme release from alveolar macrophages is different than for polymorphonuclear leukocytes.     

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.     

Chemotactic and mitogenic components of the alveolar macrophage response to particles ad neutrophil chemoattractant.

The pulmonary response to instilled particulates involves initial efflux of polymorphonuclear leukocytes (PMNs) and increased production of alveolar macrophages (AMs). The relationships of the cell migration and division components of the AM production to the initial PMN response after intrapulmonary carbon or latex administration are examined. Supernatants of lung lavages taken during early PMN migration to the alveoli promote sequential migration of PMNs, migration of monocytes, and division of pulmonary interstitial cells in normal mice. Supernatants taken during the phase of increased cell division in the lung produce no such effects, implying tht factors responsible for chemotaxis and mitosis are generated rapidly and are short-lived. A possible source is the interaction of AMs with particles, since supernatants of such incubations induce an inflammatory response in vivo. Similarly, when a synthetic chemoattractant is used, efflux of PMNs is followed by macrophages arising from migration of monocytes and from division of interstitial cells. The results suggest that particulate instillation in the lung stimulates a standard inflammatory response in which rapid generation of a factor(s) chemotactic for PMNs also attracts mononuclear cells to the alveoli. The initial efflux of cells may be explained by migration from the blood, but continued demand or replacement requires mitotic activity of precursors. For the alveolar macrophages, this includes division of cells in the pulmonary interstitium.     

Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface.     

Microscopic characterization of rabbit lung damage produced by Pseudomonas aeruginosa proteases.

The intratracheal administration of highly purified Pseudomonas aeruginosa proteases (ca. 10 to 100 microgram) elicited extensive, grossly observable rabbit lung damage by 3 h postinjection. Light and electron microscopic characterization of the lesions revealed: (i) progressive injury and necrosis of type I epithelial cells and capillary endothelial cells from 3 h to 1 day postinjection, and progressively increasing accumulations of erythrocytes, plasma proteins, fibrin, and released type II epithelial cell lamellar bodies in alveolar lumina during that time period; (ii) progressively increasing accumulations of macrophages, but not of polymorphonuclear leukocytes, in alveolar lumina from 3 h to 6 days postinjection; (iii) progressive hyperplasia of type II epithelial cells from 12 h to 4 days postinjection; (iv) progressive infiltration of alveolar septa by mononuclear inflammatory cells (interstitial pneumonitis) from 2 to 6 days postinjection; (v) no loss of alveolar septal connective tissue and no damage to pulmonary arterioles and venules; and (vi) almost normal alveolar structure by ca. 8 days postinjection. The study revealed that the intra-alveolar hemorrhage, the injury and necrosis of alveolar septal cells, and the infiltration by mononuclear cells that have been reported to occur during human pseudomonas pneumonia can also be elicited by the experimental administration of pseudomonas proteases. Thus, the results support the idea that in vivo production and activity of P. aeruginosa proteases is important, at least in part, in eliciting the lung damage characteristic of pseudomonas pneumonia.     

ALVEOLAR DESTRUCTION IN EXPERIMENTAL KLEBSIELLA PNEUMONIA

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.     

Alveolar destruction in experimental Klebsiella pneumonia

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.     

Function of h(2)o(2), myeloperoxidase, and hexose monophosphate shunt enzymes in phagocytizing cells from different species

The effect of phagocytosis on the H₂O₂ production and myeloperoxidase (MPO) activities of leukocytes from various species was investigated. The intracellular distribution of MPO, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase (6PGDH) of resting and phagocytizing guinea pig polymorphonuclear leukocytes has also been studied. Phagocytizing cells produce more H₂O₂ than the corresponding resting cells. This has been found to be true for human peripheral polymorphonuclear leukocytes, mouse peritoneal macrophage, and guinea pig and rat peritoneal polymorphonuclear leukocytes. All of these cells, except rabbit alveolar macrophages, have significant MPO activity. Generally an increased activity is noted with phagocytizing cells. Homogenization and differential centrifugation of guinea pig peritoneal polymorphonuclear leukocytes indicate that the whole homogenate and its fractions from phagocytizing cells have significantly higher MPO and NADPH oxidase activities, when compared to the corresponding fractions from the resting cells. The 27,000 × g supernatant fluid from phagocytizing cells has 6-fold more MPO and 2.5-fold more NADPH oxidase activity than similar supernatant fractions from resting cells. The enzyme 6PGDH was unaffected by phagocytosis. The relationship of these stimulated activities to the intracellular bactericidal function of the phagocytes has been discussed.     

Alveolar macrophage procoagulant activity is increased in acute hyperoxic lung injury.

Alveolar macrophage accumulation and interstitial fibrin deposition are prominent in adult respiratory distress syndrome and chronic interstitial lung diseases. The role of alveolar macrophages in the initiation of fibrin deposition and lung injury in these diseases is uncertain. Expression of procoagulant activity by these cells may provide evidence of macrophage activation and involvement in the initiation of lung fibrin deposition. An experimental model of hyperoxia-induced lung injury in rats was studied for assessment of the relationship of lung injury, fibrin deposition, and alveolar macrophage procoagulant activity. Lung injury was assessed histologically and functionally, and the accumulation of inflammatory cells was quantified by bronchoalveolar lavage. Pulmonary injury, manifested by increased capillary permeability, developed progressively during exposure to hyperoxia and was associated with significant augmentation of the procoagulant activity of alveolar macrophages early in the disease. This increase preceded the accumulation of polymorphonuclear leukocytes. Alveolar macrophage procoagulant activity had functional characteristics consistent with tissue factor. These studies provide evidence of early alveolar macrophage activation in acute hyperoxic lung injury in rats and suggest a role for procoagulant activity in the development of interstitial fibrin deposition.     

Human alveolar macrophages release a factor that inhibits phagocyte function

Human alveolar macrophages release in vitro a factor that inhibits both random migration and chemotaxis of human polymorphonuclear neutrophils (PMN). This factor is not cytotoxic and is recovered in culture supernatants of alveolar cells from most nonsmoking normal subjects. The inhibitor can be detected 30 min after cell cultures are established and is still produced after 24 h in culture. Its release was inhibited by cycloheximide. When supernatants are separated by molecular sieving (I-60 Waters HPLC column), most of the inhibitory activity is recovered in the low-molecular-weight fractions of the chromatogram (less than 1,000 D). The inhibitor has a broad spectrum of activity against known chemoattractants in that it reduces significantly the chemotaxis of PMN induced by the formyl peptide FMLP, by the complement fragment C5a, and by leukotriene B4; it also decreases the chemotactic activity associated with a monocyte-derived interleukin 1 preparation and the chemotactic activity derived from alveolar macrophage culture supernatants. The inhibitory factor is partially heat labile, is sensitive to aminopeptidase M, and is nonpolar. Both phorbol myristate acetate (PMA) and FMLP-induced superoxide release by PMN are diminished significantly in the presence of this inhibitory factor (p less than 0.01 for PMA and p less than 0.05 for FMLP). The inhibitor also reduces monocyte chemotaxis but has no effect on monocyte random migration. Finally, studies with [3H]FMLP indicate that this inhibitor does not act at the site of receptor binding on PMN. Thus, human alveolar macrophages can release in vitro both neutrophil chemotactic factors and an apparent neutrophil-inhibiting factor that may modulate positively and negatively the movement and the respiratory burst of neutrophils in the alveolar space.     

Lung injury induced by leukocytic proteases.

Human polymorphonuclear neutrophilic leukocytes (PMNs) contain large amounts of neutral proteases that can degrade elastin, collagen, proteoglycan, and basement membrane. The instillation of one of the purified enzymes (elastase) into dog lungs in vivo causes degradation of elastic fibers and other alveolar septal components and results in anatomic changes similar to those of human pulmonary emphysema. Cigarette smoking is a major risk factor associated with pulmonary emphysema in man. One mechanism for this association may be interference with the regulation of PMN elastase activity by alveolar antiproteases. This possibility is supported by the observation that the oxidizing activity of tobacco smoke inactivates alpha 1-proteinase inhibitor in vitro. Macrophages also secrete an elastolytic protease, albeit at low levels. The short-term exposure of cultured mouse macrophages to cigarette smoke augments the rate of elastase secretion by these cells. Mouse macrophage elastase is not inhibited by alpha 1-proteinase inhibitor or alpha 2-macroglobulin. This unusual property of macrophage elastase may facilitate its attack upon elastin over prolonged intervals despite very low levels of macrophage elastase production. A unified hypothesis of lung injury in pulmonary emphysema is presented, involving both PMN and macrophage elastases and the actions of cigarette smoke. (Am J Pathol 97:111--136, 1979).     

Quantitation of polymorphonuclear leukocyte migration under agarose by enzyme assay

Migration of polymorphonuclear leukocytes under agarose is widely used to assay the mobility of these cells. We modified this assay so that directed and random migration of polymorphonuclear leukocytes might be quantitated rapidly and objectively by an enzyme method. The optimal method used gelatin-agarose (0.25% gelatin; 0.875% agarose) gel layered on glass coverslips. At the end of the migration period, the leukocytes which had not migrated under the agarose were removed by aspiration and washing. The remaining leukocytes were solubilized with Triton X-100 and the myeloperoxidase activity on the coverslips determined. With appropriate standards, this activity could be used to estimate the number of polymorphonuclear leukocytes which had migrated in a random or in a directed (chemotactic) manner. Excellent correlation was observed between the myeloperoxidase method and direct microscopic counting of the number of cells that had migrated. The sensitivity of the assay could be enhanced by inducing bidirectional migration under 2 simultaneous gradients of chemotactic factor situated 180 degrees apart. This assay is objective, reproducible and combines the simplicity of assessing migration by measuring distance of the 'leading cell front' with the accuracy of individual cell counting.     

Inhaled asbestos activates a complement-dependent chemoattractant for macrophages

Pulmonary macrophages migrate to the sites where inhaled chrysotile asbestos fibers initially are deposited (i.e., surfaces of alveolar duct bifurcations). These macrophages have been shown to form a major component of an early asbestos-induced interstitial lesion in rats. In this report, we describe a potential mechanism by which macrophages are attached to these sites of fiber deposition. Chrysotile asbestos fibers used in vitro activate complement proteins in peripheral blood serum and in lavaged cell-free lung proteins. After brief inhalation of chrysotile asbestos, fluids lavaged from the lungs of exposed rats contain substantial chemotactic activity for macrophages compared to fluids from sham-exposed animals (p less than 0.01). We hypothesize that this chemotactic activity is derived from complement activated by inhaled asbestos on alveolar surfaces. This contention is supported by the following observations. Production of chemotactic activity by asbestos in vitro in serum or in lavaged lung fluids was blocked by complement inhibitors. Fractionation, by molecular sieve chromatography, of serum proteins and concentrated proteins lavaged from the lungs of asbestos-exposed rats showed that chemotactic activity was detected in the 14,000- to 18,000-dalton range. This fractionation profile is similar to C5a, the chemotactic product of complement activation. Rats treated with cobra venom factor to deplete circulating complement as well as complement-deficient mice demonstrated significantly depressed macrophage accumulation at sites of asbestos deposition. Pulmonary macrophages are the cells that form the initial inflammatory response to asbestos inhalation. Our findings support the hypothesis that asbestos fibers, and possibly other inhaled particulates, activate complement-derived chemotactic activity on alveolar surfaces. Consequently, macrophages are attracted to the alveolar duct bifurcations where inhaled asbestos fibers are deposited, and this is where the initial lesion of asbestosis is manifested.