Evaluation of free radical scavengers in studies of lymphocyte-mediated cytolysis.

Oxygen centered radicals derived from potassium superoxide (KO2) in solution were found to be toxic to P815 mastocytoma target cells. Protection of P815 cells from chemically generated radicals was mediated most efficiently by Tiron, a low molecular weight scavenger of superoxide radicals (O2-). Superoxide dismutase (SOD) and catalase also afforded protection. In vivo cytolysis of P815 target cells mediated by allogeneic lymphocytes sensitized in vivo to tumor cells was inhibited by reducing the amount of oxygen available for metabolism and by radical scavengers. Ferricytochrome c, Tiron, and phenol, all of which are relatively low molecular weight radical scavengers, inhibited cytolysis in a dose-dependent manner, while mannitol, a hydroxyl radical scavenger, did not. High molecular weight scavengers like SOD and catalase had no effect. The inhibition of cytolysis by radical scavengers was found to be independent of toxic effects on effector cells, chelation of ions, or indirect effects on prostaglandin biosynthesis. Pretreatment of either effector or target cells with Tiron had no effect on LMC. The data suggest a possible role for free radicals in molecular events leading to target cell injury by sensitized lymphocytes.

Manufacture of antithymocyte globulin (ATGAM) for clinical trials.

Methods are described for preparing large amounts of horse anti-human thymocyte globulin (ATG, ATGAM; The Upjohn Company) for clinical use. These methods have been used since 1968 to provide material for clinical trials. Characteristics of 40 lots of ATG are summarized.

An IgG thymolytic autoantibody in rats which has specificity for a subpopulation of T cells.

A cytotoxic anti-thymocyte IgG auto-antibody is present in Lewis rats which, in the presence of autologous complement, destroys (in vitro) 12-28 per cent of isologous or autologous thymocytes, a smaller number of lymph node cells and splenocytes, but not bone marrow or circulating lymphocytes. The labile cells in the thymus represent a finite subpopulation which is autologous antithymocyte antibody (ATS) sensitive and steroid resistant. The presence of the autoantibody is randomly distributed in outbred animals whereas in inbred Lewis rats, a strain in which the induction of some autoimmune reactions is under genetic control, the antibody is always present. In this strain, the susceptible T cells and the quantity of circulating autoantibody is significantly depressed during the productive phase of a T-cell mediated disease (adjuvant polyarthritis) and returns to normal after the disease becomes stabilized. There is a direct relationship between the amount of susceptible cells in the thymus and the amount of antibody in circulation, suggesting that the antibody could serve as a marker for a specific subpopulation of thymocytes which may have a regulatory influence on T-cell reactivity.

The effect of BCG on extravascular mononuclear cell accumulation in vivo.

BCG injection produces an increased accumulation of adoptively transferred 51Cr-labelled mononuclear cells at the site of inflammatory reactions in rats by two mechanisms. Systemic immunization induced a population of "activated" cells which, compared with controls, had an increased life span in the circulation after transfer and migrated in increased numbers to a chemotactic stimulus. These cells also were found in significantly greater than normal levels in the liver. In addition, local injection of BCG selectively attracted adoptively transferred mononuclear cells. These mechanisms help to explain local mononuclear cell accumulation which accompanies tumor rejection following intralesional or systemic BCG immunization.

Mechanisms by which leukocytes emigrate and induce tissue destruction.

Chronic inflammatory reactions are characterized by emigration of leukocytes to chemotactic stimuli and the subsequent release of intracellular enzymes which propogate the inflammatory reaction and cause tissue injury. The adoptive transfer of isologous 51Cr labeled leukocytes was used as a model to dissect the cellular mechanisms involved in cell accumulation to the site of immune complex or complement mediated inflammatory reactions. An intact microtubular system was necessary only for neutrophil emigration whereas cell membrane integrity was necessary for mononuclear cell but not neutrophil accumulation in vivo. The accumulation of both cell types was inhibited following phagocytosis or in the presence of corticosteroids. Release of protein polysaccharide degrading neutral protease activity from leukocytes in the presence of immune complexes was dependent upon intact microtubular activity and cell membrane integrity since agents affecting these systems inhibited enzyme release. Conversely collagenase activity released from leukocytes under the same conditions was not inhibited by these agents. Evidence will be presented indicating that the emigration of specific cell types and the release of selected enzyme systems are controlled by separate and distinct mechanistic pathways.

In vivo chemotaxis of rat leukocytes in the presence of circulating chylomicrons.

Dietary-induced chylomicronemia was produced in rats to determine its effect on the chemotactic activity of 51Cr-labelled adoptively transferred isologous leukocytes. Rats fed a low protein high fat diet for 2 months had normal total plasma cholesterol and triglyceride levels, but increased amounts of chylomicrons. Circulating neutrophils and monocytes from animals on a normal diet were able to phagocytose chylomicrons from plasma of those animals on the special diet. Peritoneal exudate cells from the latter animals contained intracellular chylomicrons demonstratable both histologically and biochemically. Neutrophils and mononuclear cells from normal or special fed animals, when transferred to chylomicronemic recipients, had a reduced ability to accumulate at the site of a complement-dependent inflammatory reaction but circulated normally. The defective chemotactic activity observed could be duplicated when cells were allowed to ingest aggregated protein instead of chylomicrons. It was concluded that circulating leukocytes ingest chylomicrons from the plasma with a resultant reduction in their chemotactic activity. The results were discussed in relation to the increased incidence of infection in diabetes characterized by Type I and V hyperlipidemias. It was also suggested that phagocytosis of particulate substances by entrapped leukocytes in atherosclerotic lesions would induce phagocytic enzyme release which could contribute to the eventual destruction of the vascular wall.

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.

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.

An analysis of the specificity in pharmacological inhibition of the passive cutaneous anaphylaxis reaction in mice and rats.

An antiserum obtained from mice, immunized to produce an antiovalbumin antibody of the IgE type, was employed in a 48-hour passive cutaneous anaphylaxis (PCA) reaction in both mice and rats. The antiserum contained an antibody which, "fixed" to skin for at least 6 days, was heat labile and eluted from diethylaminoethyl cellulose in the reagin peak. In both rats and mice, the PCA reaction was mediated by a combination of histamine and serotonin and was inhibited by specific antagonists. Various drugs were tested for inhibition of the PCA reaction in recipients also injected with compound 48/80 and histamine. Drugs which have been reported to cause an increase in intracellular cyclic adenosine monophosphate levels [prostaglandins (PG) E1 and E2 and theophylline] all selectively inhibited the PCA reaction at low doses. By varying the length of time of drug administration prior to antigen challenge, the pharmacological half-life of PGE1 was determined to be approximately 9 minutes. At high doses, theophylline also inhibited the 48/80 reaction, and PGE1 inhibited all three reactions, whereas PGE2 only inhibited PCA. Disodium cromoglycate, when given to rats, inhibited only the PCA reaction without effect on the 48/80 or histamine wheal. It was totally ineffective on any parameter measured in the mouse. It is suggested that the PCA reaction in the rodent is induced by an IgE-like antibody and mediator release is, to some extent, sensitive to intracellular levels of cyclic adenosine monophosphate. Analysis of the specificity of drug activity depends upon dose-response studies, species differences and consideration of nonspecific systemic effects.