A rapid microtiter plate method for the detection of lysozyme release from human neutrophils.

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental arthropathy induced in rhesus monkeys and DBA/1 mice by a novel method: intraperitoneal implantation of type II collagen adsorbed onto nitrocellulose filters.

A novel method which avoids the use of complete Freund's adjuvant (which can be arthritogenic) has been used to induce collagen II arthritis in both primates and mice. A solution of bovine type II collagen was dried onto nitrocellulose filters and implanted in the peritoneal cavity of experimental animals. Primate and mouse joints were scored by clinical as well as gross and microscopic parameters. The polyarthritis that developed in both rhesus monkeys (Macaca mulatta) and DBA/1 LAC J mice was characterized by synovial cell proliferation and endothelial cell hyperplasia, and by a perivascular mononuclear cell infiltrate of the synovium. Primates were analyzed further for anti-type II collagen antibody titers and delayed type hypersensitivity to type II collagen. Anti-type II collagen serum titers appeared to be unrelated to the disease pathology; the primates did not display delayed-type hypersensitivity to type II collagen. Control monkeys and mice implanted with collagen-free nitrocellulose filters were normal upon clinical and histopathological analysis. This protocol offers the advantage of the induction of arthritis due solely to immunization with antigen.

Experimental arthropathy induced in rhesus monkeys (Macaca mulatta) by intradermal immunization with native bovine type II collagen.

Over a 6-month time course, polyarticular arthritis was induced in 7 male rhesus monkeys by 3 intradermal injections of bovine type II collagen emulsified in complete Freund's adjuvant, followed later by 2 intradermal injections of type II collagen in incomplete Freund's adjuvant. All animals exhibited delayed-type hypersensitivity to type II collagen by skin test and had serum anti-type II collagen titers of greater than 10,000 (at 1 month) and 20,000 to 160,000 (at 6 months) by enzyme-linked immunosorbent assay. Gross joint changes were observed in 6 of 7 monkeys, especially in the knee and elbow; synovial cell hyperplasia, increased vascularization and a focal mononuclear cell infiltrate were the most frequent findings. Chronic arthritis with destructive cartilage lesions was most prominent in the phalangeal joints of the hands (7 of 7 animals). Microscopically, these changes consisted of fibrosis of the synovium with increased vascularization, villous synovial membrane hyperplasia and focal mononuclear cell infiltration, as well as fibrous metaplasia of the articular cartilage adjacent to pannus formations. Also evident was a loss of safranin O staining intensity in the cartilage and loss of continuity of the articular surface. The 7 control monkeys (received Freund's adjuvant without collagen) were delayed-type hypersensitivity-negative, had no serum anti-type II collagen antibodies, and had grossly and microscopically normal joints. This primate model resembles collagen-induced arthritis seen in rodents and, to some degree, human rheumatoid arthritis.

Chemical identification of a prostaglandin-induced T suppressor (PITS).

Prostaglandin E1 is known to block the activation and function of immunocompetent cells. Previous studies from this laboratory have established that one pathway where prostaglandin E1 acts involves the stimulation of a glass-wool adherent T-suppressor cell. Prostaglandin stimulates this suppressor cell to release a suppressor lymphokine(s), termed the prostaglandin-induced T cell-derived suppressor (PITS). We report here the further characterization of this factor using high-pressure liquid chromatographic analyses, precursor labeling studies, and bioanalysis, all of which indicate that this factor contains a leukotriene.

Purification and biochemical analysis of antigen-specific suppressor factors obtained from the supernatant, membrane, or cytosol of a T cell hybridoma.

A suppressor factor (TsF) specific for the synthetic terpolymer L-glutamic acid60-L-alanine30-L-tyrosine 10(GAT) produced by the T cell hybridoma 342B1.11 has been purified to apparent chemical homogeneity. This TsF was found in the cell culture supernatant, was associated with the cell membrane fraction, and was found in the cytosol. The supernatant form of TsF occurs as a single polypeptide chain of 29 to 30 k m.w., which easily forms aggregates of 65 k m.w. The membrane-associated form of TsF exists primarily as 65 k m.w., the cytosol TsF exists as both 65 and 29 to 30 k m.w. Amino acid analysis of each source of TsF shows the identical mole percent of amino acids, suggesting that all forms of TsF are derived from a single peptide of 29-30 k m.w. Analysis of the supernatant TsF by electroblot with the use of the anti-I-Js antisera (H-2s is the haplotype of the spleen cell donor) indicates that the polypeptide bears an I-J determinant, as do other TsF of this type.