Identification of neutrophil chemotactic factors in bronchoalveolar lavage fluid of asthmatic patients.

BACKGROUND: Although neutrophils have been implicated in bronchial asthma, the mechanism(s) which bring these cells into the airways is poorly understood. OBJECTIVE: To investigate the presence and identity of neutrophil chemotactic factors in bronchoalveolar lavage (BAL) fluid from atopic asthmatic subjects. METHOD: BAL fluid was obtained from 13 subjects (seven asthmatics and six normals), aged 19 to 60 yr, at bronchoscopy. Separation of neutrophil chemotactic activity (NCA) was achieved by FPLC cation exchange chromatography. Fractions were collected and assayed for chemotaxis in multiwell micro-chemotaxes chambers using polycarbonate filters, for the complement peptide C5a/C5a des Arg by radioimmunoassay (RIA) and for interleukin-8 (IL-8) by ELISA. RESULTS: NCA was found in FPLC fractions of BAL samples in four out of seven asthmatics and each of these subjects had at least three similar peaks of NCA. The major peak of NCA was found to contain immunoreactive C5a/C5a des Arg and chemotaxis. In response to this NCA could be blocked by desensitization of the neutrophils with recombinant C5a. Purified serum derived C5a/C5a des Arg was found to have altered chromatographic properties when added to BAL fluid; this suggested that BAL fluid contained proteins which interacted with the C5a/C5a des Arg. Immunoreactive IL-8 (iIL-8) was also detected but its concentration or chemical form was insufficient to induce neutrophil chemotaxis. CONCLUSION: This study demonstrates that bronchial asthmatic lavage fluid contains C5a/ C5a des/Arg and IL-8, together with other as yet unidentified factors which may contribute to neutrophil recruitment in this disease.

Surprisingly high levels of anaphylatoxin C5a des Arg are extractable from psoriatic scales.

Water-soluble extracts from psoriatic scales and normal human skin were prepared using either phosphate-buffered saline, pH 7.2, or 0.1 M carbonate buffer, pH 10.8. Anaphylatoxin C5a des Arg was quantified using a novel sandwich enzyme-linked immunosorbent assay (ELISA) using neoepitope-specific monoclonal antibodies. Alkali was about five to eight times more efficient than PBS in extracting C5a des Arg from scales, probably via dissociation of bound C5a des Arg. C5a des Arg concentration in scales from three patients suffering from psoriasis vulgaris varied between 2.5 and 4.6 ng/mg scale. No C5a des Arg was detectable in normal skin extracts. The biological activity of alkali-extractable C5a des Arg, i.e. chemotaxis, was preserved. The concentration of C5a des Arg relative to the concentration of albumin was taken as a parameter of the degree of complement activation in the psoriatic lesions, and was found to be more than six times higher than values attained in serum after maximum complement activation by zymosan. We conclude that complement activation may play a quantitatively important role in the inflammatory process in psoriasis.

Three-dimensional structure of porcine C5adesArg from 1H nuclear magnetic resonance data.

Two-dimensional nuclear magnetic resonance spectra of porcine C5adesArg (73 residues) have been used to construct a list of 34 hydrogen bonds, 27 dihedral angle constraints, and 151 distance constraints, derived from nuclear Overhauser effect data. These constraints were used in restrained molecular dynamics calculations on residues 1-65 of C5a, starting from a folded structure modeled on the crystal structure of a homologous protein, C3a. Forty-one structures have been calculated, which fall into three similar families with few violations of the imposed constraints. Structures in the most populated family have a root-mean-square deviation from the average structure of 1.02 A for the C alpha atoms, with good definition of the internal residues. There is good agreement between the calculated structures and other nuclear magnetic resonance data. The structure is very similar to that recently reported for human C5a [Zuiderweg et al. (1989) Biochemistry 28, 172-185]. Some biological implications of these structures are discussed.