Alveolar macrophage-derived chemotactic factor: kinetics of in vitro production and partial characterization.

Abstract

A B S T R A C T Alveolar macrophages are the initial phagocytic cells that encounter foreign material and particulates deposited in the terminal airways. We have examined a mechanism by which these cells, after phagocytic challenge, may control or amplify the inflammatory response in lung parenchyma. Normal human alveolar macrophages (AM) were studied from eight subjects. With in vitro culture, AM produced and released two substances into culture media which have potent chemoattractant activity for blood polymorphonuclear granulocytes(PMN) and negligible activity for mononuclear cells. Release of these factors is maximally stimulated by aggregated human immunoglobulin (Ig)G or zymosan particles; however, simple adhesion of the macrophages to plastic surfaces is also sufficient to stimulate release of these chemotactic substances.The larger substance (10,000 daltons) is immunologically distinct from C5a and interacts with a different PMN membrane receptor than that known to exist for formyl-methionyl-leucyl-phenylalanine. Its chemotactic activity is sensitive to the enzymatic effect of trypsin. Although producing a single elution peak on gelfiltration chromatography, electrofocusing in polyacrylamide gels yielded five peaks of radioactivity. Chemotactic activity was localized to a fraction with a pI = 5.0. The smaller molecular weight substance has been less well characterized. Thus, the human AM can produce at least two factors which attract PMN and this capability may augment the local inflammatory response in the lung.

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan.

“…Merrill and colleagues (48) found neutrophil chemotaxins with molecular weights of about 10,000 and < 1,000 daltons in 24-h culture fluids of human AM. Based on our data, the larger molecule was probably NAP-1.…”

Complement Activation in Cystic Fibrosis Respiratory Fluids: in Vivo and in Vitro Generation of C5a and Chemotactic Activity

“…These normal controls had no history of previous Pseudomonas infections and had not recently experienced respiratory infections. Blood was obtained from these 14 experimental subjects, serum was immediately separated by centrifugation at 4" C and stored at -78" C. Lung lavage to obtain respiratory secretions from CF patients and control subjects was performed and the fluids were processed as previously reported (8)(9)(10). The volume of the lung lavage fluid from each subject was recorded and split into two portions: to one aliquot both NaN3 and EDTA were added to final concentrations of 0.01% (w/v) and 10 mM, respectively.…”

“…PM were obtained from healthy normal nonsmoking volunteers who underwent transoral flexible fiberoptic bronchoscopy and lung lavage as described above. The lavage fluid cell pellet (93% macrophages, 7% lymphocytes) was then processed in a standard fashion for short-term cell cultures (2,8,10,26). Sixteen hours later, culture media was removed and replaced with 1 ml aliquots of CF lavage fluid ( n = 5), lavage fluid specimens from normal volunteers ( n = 4), and CF BAL fluid specimens with added PMSF (1 mM) or EDTA (50 mM).…”

“…Pulmonary macrophages possess C5 polypeptides attached to surface membranes (Robbins RA, Russ WD, Thomas KR, Rasmussen J, Kay HD, unpublished data) and when stimulated are known to release chemotactic substances for neutrophils (10). In order to investigate the role PM might play in the generation of C5a anaphylatoxins normal human PM were cultured in the presence of CF and normal BAL fluids and then supernatant fluids were assayed for chemotactic activity.…”

Antibody to Multiple Mucoid Strains of Pseudomonas aeruginosa in Patients with Cystic Fibrosis, Measured by an Enzyme-Linked Immunosorbent Assay

“…Bronchial washings were performed on eight patients with various diagnoses. They were clinically stable at the time of the lavage and specimens were processed as published by Merrill et al (13). Total protein was determined by Coomassie brilliant blue absorbance (14) and was 0.8 to 1.6 mg/ml after concentration 10-to 20-fold using positive pressure ultrafiltration (Diaflo YM5 membrane, Amicon Corp., Scientific Systems Division., Lexington, MA).…”