Arnold L. Oronsky

Breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates from guinea pig, rabbit, and human

Abstract Degradation of the noncollagenous protein-mucopolysaccharide matrix of rabbit ear cartilage in vitro by various enzyme preparations is reported. Cartilage matrix degradation was quantitated by measuring the release of soluble, 35S-labeled, anionic material from cartilage that was previously labeled in vivo with Na235SO4. The release of 35S was specific for chondromucoprotein degradation, as evidenced by the parallel release of mucopolysaccharide split products, precipitation of 35S with cetyltrimethyl ammonium bromide and enzyme specificity. Autolytic breakdown of chondromucoprotein proceeded predominantly at pH 5.0 and to a very limited extent at pH 7.4 during an 18-hr incubation at 37°C. Trypsin, chymotrypsin, and elastase provoked chondromucoprotein degradation at pH 7.4, whereas hyaluronidase was more active at pH 5.0. Purified bacterial collagenase did not promote significant breakdown of the noncollagenous portion of cartilate matrix at neutral or acid pH. Lysosome granule lysates of guinea pig and rabbit peritoneal polymor-phonuclear leukocytes were capable of degrading the chondromucoprotein of cartilage matrix at pH 5.0 but not at pH 7.4. Conversely, lysates of granules from human leukocytes damaged cartilage matrix at pH 7.4 but not at pH 5.0. Gold sodium thiomalate (10−4M) and human serum (10%) inhibited the degradation of guinea pig, matrix provoked by autolysis and by the leukocyte granuleslysates of guinea pig, rabbit and human. The demonstration in human leukocyte granules of neutral protease(s) that damage the noncollagenous chondromucoprotein matrix of cartilage and the finding that gold, an antirheumatic drug, inhibits this deleterious effect, may have a significant bearing on the pathology of connective tissue disease.

CONNECTIVE TISSUE‐DEGRADING ENZYMES OF HUMAN LEUKOCYTES

Human leukocytes, when exposed to aggregated human gamma-globulin (AHGG) or immune complexes (isolated from RA synovial fluid) fixed to a cartilagenous surface, release neutral proteases that degrade the extracellular matrix of cartilage. The chondromucoprotein destruction by these proteases is suppressed by a variety of synovial fluids but is not susceptible to inhibition by trypsin, chymotrypsin, elastase inhibitors, or a combination of these agents. The inhibitory effect of synovial fluid can be reversed in the presence of increasing enzyme concentrations. Intact viable human polymorphonuclear leukocytes in the presence of AHGG also release a collagenase precursor that can be activated by limited proteolysis with trypsin or RA synovial fluids. Enzyme release (neutral proteases) by phagocytosing cells is inhibited by the antiinflammatory agents phenylbutazone and colchicine; these agents do not affect release of the collagenase precursor. However, the latent collagenase release is susceptible to inhibition when leukocytes are preincubated (prior to exposure to AHGG) with inhibitors of protein synthesis. Under these conditions, neutral protease release is unaffected.

Phagocytic release of human leukocyte neutral proteases: Analysis of cartilage degradation products

Abstract Human polymorphonuclear leukocyte neutral proteases (HLNP) released during the process of phagocytosis of aggregated human gamma globulin were tested for their ability to degrade intact rabbit ear cartilage. Using 35 S-labeled cartilage as substrate, HLNP derived from 45 × 10 7 cells released about 45% of the total incorporated 35 S. DE-52 chromatography of incubation supernatants revealed a single 35 S peak associated with minimal quantities of peptide or protein material as estimated by absorbance at OD 230 + 280 nm . Analytical ultracentrifugation gave a molecular weight of 51,800. Incubation of cartilage with excess α-chymotrypsin released 35 S-containing protein and peptide elements ( M r 79,400). Therefore, degradation, of the proteoglycans of intact cartilage by HLNP is more extensive than that noted with bovine pancreas α-chymotrypsin. The products of HLNP and α-chymotrypsin digestion of cartilage contained chondroitin sulfates A and/or C since both materials (after column chromatography) were sensitive to chrondroitinase ABC and AC digestion. Collagenolytic activity of HLNP is minimal compared to proteolytic activity as evidenced by the lack of hydroxyproline containing peptides released from cartilage during enzyme incubation. It is suggested that HNLP incubated with intact cartilage may serve as a relevant model in the search for new agents which could combat enzyme-mediated cartilage destruction.

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

Abstract Hepatic fibrosis in rats was induced by chronic subcutaneous treatment with carbon tetrachloride. The amount of collagen and the specific activity of prolyl hydroxylase were greatly enhanced. On treatment of these fibrotic animals with dl -3,4-dehydroproline (100 mg/kg) for 5 consecutive days, the specific activity of liver prolyl hydroxylase was greatly decreased and approached the activity observed in the liver of normal rats. Although carbon tetrachloride did not affect prolyl hydroxylase activity of the lung, administration of dehydroproline caused a significant decrease in enzyme activity. Collagen extracted from normal and fibrotic rat livers was analyzed by chromatography on carboxymethylcellulose. Normal rat liver contains α 1 and α 2 chains with Chromatographic properties similar to Type I collagen of calf skin. Fibrotic liver contains Type I collagen as well as collagen with Chromatographic properties similar to Type III. The increase in the latter type of collagen is more pronounced in the liver of fibrotic animals.

PHYSIOLOGIC AND PHARMACOLOGIC ALTERATIONS OF RAT LEUKOCYTE CHEMOTAXIS (Cx) IN VIVO

The method of adoptively transferring 51Cr-labeled rat leukocytes iv to isologous recipients was used to quantitate the extravascular accumulation of specific cell types at the site of inflammation induced by local injection of various phlogistic agents. Experiments were designed to determine whether cellular accumulation could be modified at the level of the chemotactic factor, by serum components, or by alteration of the responding cell itself. The results indicated a selective attraction of mononuclear cells to the local injection site of BCG and of neutrophils to the injection site of aggregated but not monometric gamma-globulin. Thus, leukocytic accumulation was found to be dependent upon the local generation of specific reactants that were particular to the agent employed. Leukocytic accumulation could also be modified by serum factors. Cellular accumulation was inhibited when leukocytes were exposed to serum that contained phagocytosable particles or after phagocytosis in vitro prior to adoptive transfer. Chemotaxis of lymphocytes could be induced by their preincubation with sera from adjuvant arthritic animals. These observations were confirmed by in vitro studies and by the finding that 6 days after adjuvant injection , lymphocytes but not mononuclear cells accumulated at the noninjected extremity. In the final series of experiments, it was shown that BCG immunization was capable of inducing a unique population of peritoneal mononuclear cells that after adoptive transfer had an enhanced capacity to remain in the circulation, which, in turn, resulted in a functional increase in their accumulation at an inflammatory reaction site. In conclusion, these studies indicated that the chemotactic activity of adoptively transferred cells can be modified by changes in the chemotactic stimuli, can be enhanced or depressed by serum factors, and is a function of the physiologic capability of the cell population employed.

The Selective Inhibition of Secondary IgE Antibody Response in Mice by Dibutyryl Adenosine 3′,5′-Cyclic Monophosphoric Acid and Theophylline

Summary The effect of various agents on the in vivo anamnestic IgE and IgG responses of mice to alum suspensions of ovalbumin was determined. Dibutyryl cAMP and theophylline inhibited IgE but not IgG synthesis, whereas other immunosuppressive drugs inhibited both antibody classes equally. Previous reports have suggested separate pathways for the synthesis of IgG and IgE, and this report suggests that the IgE response can be selectively inhibited.

Chapter 6: Pulmonary and Anti-allergy Drugs

Publisher Summary There are two broad categories of allergic reaction: immediate and delayed hypersensitivity. Delayed responses include allergies to poison ivy, drugs, chemicals, etc., while immediate hypersensitivity includes syndromes such as allergicrhinitis, uticaria, and asthma. Bronchial asthma may be classified into intrinsic (not clearly associated with a known imunologic mechanism) and extrinsic or allergic asthma that may be mediated by the union of antigen with specialized immunoglobulins (IgE). Theories concerning the pathogenesis of extrinsic asthma have been advanced, including decreased β-adrenoreceptor activity, “increased α-adrenergic activity” and changes in reflex cholinergic mechanisms. The pathogenesis may be divided into six categories. Experiments on rat and guinea pig include use of Beta-Adrenoreceptor Stimulants, α-Adrenoreceptor Antagonists, Anti-cholinergics, Prostaglandins, Corticosteroids, Phosphodiesterase Inhibitors, and peptides. Phannacokinetics of dihydroxypropyl theophylline (dyphylline) by intramuscular and by oral administration (10 mg/kg) indicate a half-life of two hours and ready absorption independent of route. A combination of theophylline—ephedrine has been shown to be no more effective than theophylline alone and also to cue GII side, insomnia and restlessness not found with the same dose of the individual drugs.

Pulmonary and Anti-Allergy Drugs

Publisher Summary The interaction of cell bound immunoglobulins (Ig) with antigen and haptens leads to the release of mediators of immediate hypersensitivity from these stimulated cells. IgE appears to interact with a specific plasma membrane receptor on the target cell. Characterization of the cellular receptor for IgE is determined using rat peritoneal mast cells and rat basophilic leukemia cells and indicates that it has a molecular weight of 60,000. In carbuterol, a series of compounds of general structure substitute in the benzyl group are claimed to be 10 x 5 in potency as bronchodilators with higher bronchoselectivity. A series of mono and diesters of N-t-butylarterenol shows that the monoesters have a moderate degree of activity with rapid onset and two hours duration of action. Structure–activity relationships for a series of sympathomimetic arnines, having a carbostyril moiety, are discussed in this chapter. Detailed pharmacology, toxicology, pharmacokinetics, and metabolite patterns of clenbuterol in rat, rabbit, dog, and man are also discussed in the chapter. In clenbuterol, synthesis, structure activity relationships, and pharmacology of a series of bronchospasmolytic, 0-phenylethylaminoalkylxanthines are selected for clinical trial. Corricosreroids–beclomethasone dipropionate aerosol was used in the treatment of chronic asthma, particularly in children.