Comparative Investigations of Various Immunoregulatory Substances in the Delayed Type Hypersensitivity Test of the Mouse

Abstract

The substances D-penicillamine, auranofin, chloroquine, levamisole, BM 41.332, azimexone, bestatin, methisoprinol (inosiplex), thymosine (fraction 51, indomethacin and cyclophosphamide were examined comparatively in the delayed type hypersensitivity test after oxazolone sensitisation in mice. It was found, that only the basal antirheumatic drugs D-penicillamine, auranofin, chloroquine and levamisole and also BM 41.332 led to a potentiation of the DTH reactions. Methisoprinol, bestatin, azimexone, thymosine fraction 5 and indomethacin had no effect on the DTH, whilst the immunosuppressant cyclophosphamide led to an inhibition of the DTH reaction.

It is concluded that this pharmacological model is suitable for screening of new basal drugs for rheumatoid arthritis.     

Self-Reactive Delayed Type Hypersensitivity Induced in Mice by Syngeneic Lymphoblasts

X-irradiated or normal A mice injected with syngeneic concanavalin A-induced lymphoblasts (syn-Con A blasts) developed inflammatory responses in their footpads 24 to 72 h after the injection of syngeneic lipopolysaccharide-induced lymphoblasts (syn-LPS blasts) into these tissues. This response was designated syngeneic delayed type hypersensitivity (syn-DTH). The Con A blast extracts contain small (apparent MW of 6000-7000) and large (apparent MW of 160,000-175,000) syn-DTH-stimulating antigens, which are found in the total volume (low molecular weight fraction) and the void volume (high molecular weight fraction), respectively, of AcA 44 gel filtrations of this extract. The small and large antigens exhibit different immunological properties. The small antigen of A mouse lymphoblasts induced syn-DTH in X-irradiated (250 rad) mice but not in normal mice, and this immunological activity was elicited with syngeneic but not allogeneic lymphoblasts. The syn-DTH induced with the small antigen was inhibited by Lyt-1+2+, I-Jk+ suppressor T cells or a factor extracted from these cells. In contrast to the small antigen, the large antigen of A mouse lymphoblasts induced syn-DTH in both normal and X-irradiated mice, and this immunological activity was elicited by both syngeneic and allogeneic LPS lymphoblasts. The small and large antigens do not immunologically cross-react, but their immunogenicity is not affected by ultraviolet irradiation, indicating that the immune response against both of them is relatively class II-independent. The possibility that the cellular autoanti-lymphoblast response observed in our studies is in fact a mechanism that down-regulates the lymphoblast activity and thus suppresses the immune response is discussed.     

Self-Reactive Delayed Type Hypersensitivity Induced in Mice by Syngeneic Lymphoblasts

X-irradiated (250 rad) or normal A mice injected with syngeneic concanavalin A-induced lymphoblasts (syn-Con A blasts) developed an inflammatory response in their footpads 24 to 72 h after injection of syngeneic lipopolysaccharide-induced lymphoblasts (syn-LPS blasts) into these tissues. This immunological activity was designated syngeneic delayed type hypersensitivity (syn-DTH), because T cells transferred the response to naive recipients. Analysis on Ultrogel or Sephadex G-50 columns revealed that a Con A-blast extract contains two syn-DTH-stimulating antigens: a small antigen (6000-7000) and a large antigen (apparent MW of 160,000-175,000). This conclusion held true even when protease inhibitors were included in the fractionation procedure. The approximate molecular weights of these antigens estimated by the gel filtrations were confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The large lymphoblast syn-DTH-stimulating antigen contains carbohydrate residues but not products of the H-2 genetic region. The small antigen does not contain sugar moieties, but it expresses affinity to anti-H-2Dd monoclonal antibody. The immune response to the small antigen but not to the large antigen is genetically restricted at both the induction and the elicitation phases of the DTH. A strain of mice immunized with the small antigen generated syn-DTH after challenge with lymphoblasts of B10.T (6R) mice, which share the H-2Dd subregion with A mice but not the H-2K or the H-2I subregions. Fast protein liquid chromatography of the small antigen yielded a purified material which appeared as a single band after Coomassie staining of its gel electrophoresis.     

Delayed-Type Hypersensitivity in the Mouse

Delayed-type hypersensitivity (DTH) to Salmonella polymerized flagellin (POL) was transferred to normal and lethally irradiated CBA mice by POL-activated thymus-derived (T) cells or by lymphoid cells rich in T cells from POL-primed donors, but not by POL-activated bone marrow-derived (B) cells or by serum from POL-primed donors. It was therefore concluded that T cells play a key role in the initiation of DTH reactions. T cells activated by POL of one specificity were equally able to transfer DTH to POL of the same or of another specificity. DTH to haemocyanin (HCY) ( Jasus lalandii ) was transferred only by HCY-activated T cells. Two interpretations of these findings are discussed: 1) that T cells may be less restricted in specificity than B cells, and 2) that T cells recognize a common antigenic determinant (carrier determinant) on the polymerized flagellins of Salmonella spp , which is not recognized by B cells.     

Delayed-Type Hypersensitivity in the Mouse

The ability of thymus-derived (T) cells activated by polymerized flagellin (POL) from Salmonella adelaide to transfer anti-POL delayed-type hypersensitivity (DTH) to normal mice was largely abrogated by pretreating the cells with anti-Θ serum and complement, and completely abrogated by pretreating the cells with POL labelled with ¹²⁵I-iodine to hight specific activity. The thymocyte precursors of POL-activated T cells were also inactivated by treatment with ¹²⁵I-POL. T cells activated by POL of one specificity were completely inactivated by ¹²⁵I-POL of the same or of anothei specificity but not at all by ¹²⁵I-labelIed haemocyanin (Jasus lalandii) . Preincubation of POL-activated T cells with rabbit anti-mouse light chain globulin prevented their subsequent inactivation by ¹²⁵I-POL but did not reduce their ability to transfer anti-POL DTH. The significance of these findings is discussed.     

Delayed-Type Hypersensitivity in the Mouse

A simple reproducible footpad assay of delayed-type hypersensitivity (DTH) in CBA, C3H, and Bagg mice to Salmonella adelaide flagellin and its derivatives is described. DTH reactions were of delayed onset (12-24 hours), long duration (3-4 days), and characterized histologically by extensive infiltration of the hypodermis and muscle with mononuclear cells. Immediate (0-3 hours) and late (36-48 hours) Arthus reactions were also observed in certain instances but did not preclude detection of the 24-hour DTH reaction. Maximal DTH reactivity was induced by microgram doses of monomeric (MON) or polymerized (POL) flagellin in saline, 2-8 days after primary challenge. POL elicited greater DTH reactions than MON, presumably because of its larger size.     

Alterations in Murine Delayed Type Hypersensitivity Responses by Delta-8-THC and Cannabinol

The ability of cannabinol and delta-8-tetrahydrocannabinol (delta-8-THC), two cannabinoid mihuana components, to msdify delayed-type hypersensitivity (DTH) to sheep red blood cells (SPEC) was investigated in mice. Reduction of DTH reactivity by cannabinol required multiple, daily, postimnunization drug administration. Neither multiple preimnunization dosings nor a single postirrununization dosing was effective. Delta-8-THC produced Werate (13-33%) suppression of DTH reactivity only upn multiple, preimnunization treatments, whereas postimnunization administration only delayed peak DTH reactivity 24 hours Without altering the absolute response level. These results indicate that delta-8-THC and cannabinol possess slight to msderate activity in the suppression of DTH.     

Analysis of Peripheral Blood Lymphocyte Subsets,NK Cells,and Delayed Type Hypersensitivity Skin Test in Patients With Premature Ovarian Failure

PROBLEM : Premature ovarian failure (POF) probably belongs to the group of autoimmune endocrinopathies. Cell-mediated immune parameters were investigated. Sex steroids have a profound effect on the immune system. POF patients and postmenopausal control women (PM) were tested with or without estrogen substitution. METHOD : A novel FACS analysis system (using double labeling techniques) was used in 30 patients with POF to enumerate the subjects of peripheral blood lymphocytes and NK cells. Eighteen PM women and 30 healthy men and women served as controls. We also tested the delayed type hypersensitivity skin test (DTH) toward Candida in the POF patient group to be informed on their cell-mediated immune function. RESULTS : The numbers of blood lymphocytes, CD3⁺, CD4⁺ and CD8⁺ T cells, were not abnormal in POF patients. However, HLA-DR⁺ T cells were increased in POF patients and in PM women (P<0.05). These elevated numbers were partially reversible by estrogen substitution. The number of CD19⁺ cells (B cells) was elevated, whereas CD3^?/CD16⁺/CD56⁺ cells (NK cells) were decreased in POF patients (P<0.05), irrespective of estrogen substitution. DTH skin tests toward 0.1% Candidin (0.1 ml intradermal injection) were negative in 11 out of 20 tested POF patients, compared to only 2 out of 10 tested controls (P<0.05). CONCLUSION : POF patients show numerous immune cell abnormalities. These abnormalities were only partially due to estrogen deficiency. We hypothesize that these abnormalities either lead to ovarian autoimmunity or may have direct effects on the ovarian function.     

Eosinophils in Delayed Hypersensitivity Skin Reaction Sites

Guinea pigs immunized with egg albumin or an azobenzene arsanate conjugate of acetyl-tyrosine (ABA-tyr) in complete or incomplete Freund's adjuvant can develop eosinophil-containing delayed hypersensitivity skin reactions when challenged with specific antigen. Retest, achieved by the re-injection of antigen at the site of a prior skin test, leads to an accelerated reaction which may contain increased numbers of eosinophils. ABA-tyr which induces delayed hypersensitivity without antibody formation, dissociates the two components of the retest reaction; that antigen leads to an accelerated reaction, but one with only small numbers of eosinophils.     

Human Leukocyte Cyclic AMP and Cyclic GMP Levels during Chemotaxis in Delayed Type Hypersensitivity

Ten nickel-allergy patients and six healthy control subjects participated in a study of the morphology kinetics and evolution of the AMP and GMP concentration of migrated leukocytes, using an improved skin chambet technique. Also studied was the effect of nickel exposure in the patients Nickel exposure had a specific effect on the morphology from the 24th hour to the end of the 48 h observation period, with a significant increase in the percentages of basophils, eosinophils and lymphocytes and a decrease of neutrophils. A Significantly increased leukocyte migration rate (LMR) was observed from the 27th to 39th hour in six of the allergic patients exposed to nickel. There were no specific permanent changes in cAMP and cGMP concentration during nickel exposure. The control chambers of the allergy patients and health control had identical leukocyte morphology, LMR and leukocyte concentrations of cAMP and cGMP. However no correlations were found between LMR cAMP and cGMP in the eczema patients throughout the observation period.     

Delayed Hypersensitivity Skin Testing of 150 Volunteers

We studied skin test reactivity to five commonly used antigens by testing 150 healthy adults. The delayed hypersensitivity (DH) skin test is widely used to assess the immune status of patients. The battery of antigens suitable for use may vary in different countries, but the reactivity to the antigens in our population did not differ remarkably from reports of other authors. The reactivity rates were: candida 32.7%, mumps 86%, streptokinase-streptodornase (SK-SD) 70%, trichophyton 0% and tuberculin 58.7%. Sixteen of the subjects were retested after 2 weeks. Only eight of them showed unchanged reactions to all five antigens. Specific IgG antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA) against each of the five antigens in the serum of 42 subjects before and after testing showed great inter-individual variation. The antibody concentration did not correlate with the DH skin test results, but the testing itself increased the production of anti-mumps- and anti-SK-SD-antibodies.     

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an in vivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice immunized with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 hr after immunized mice received an intraperitoneal injection of 200 μg of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an in vivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice immunized with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 hr after immunized mice received an intraperitoneal injection of 200 μg of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

Relationship of Antimicrobial Cellular Immunity to Delayed Hypersensitivity in Listeriosis

The relationship of antimicrobial cellular immunity to delayed hypersensitivity (DH) was studied in mice antigenically stimulated by living Listeria monocytogenes confined to diffusion chambers in peritoneal cavities or by subcutaneous inoculation of sublethal doses of the organism. Mice showed DH reactions when tested 6 days after inoculation, and reactions were positive for at least 90 days in some mice. DH also became established when the mice were stimulated by antigens diffusing from peritoneal chambers containing viable Listeria. Mice were categorized as DH positive or DH negative if they developed more or less than a 5% increase in foot volume 24 h after the injection of Listeria antigen. Some antigenically stimulated mice did not elicit the DH reaction. Consequently, the animals were arranged as immunized groups (DH positive and DH negative) and Listeria chamber implant groups (DH positive and DH negative). When challenged with L. monocytogenes, all four groups were significantly resistant as compared with controls. Thus, the in vivo tests for immunity and DH did not show direct correlation. The results suggested that antimicrobial cellular immunity can occur as a phenomenon independent of DH. Evidence for antimicrobial cellular immunity as the principle mechanism of resistance in murine listeriosis is discussed with consideration for possible heterogeneity of function by thymus-derived lymphocytes.     

In Vitro Correlates of Delayed Hypersensitivity in Man: Ambiguity of Polymorphonuclear Neutrophils as Indicator Cells in Leukocyte Migration Test

Delayed cutaneous hypersensitivity (DCH) of 12 normal adult subjects to purified protein derivative (PPD) of Mycobacterium tuberculosis, streptococcal streptokinase-streptodornase (SK-SD), and Candida albicans Dermatophytin O (DO) was assayed in vivo by skin testing and compared with such in vitro correlates of cellular immunity as lymphocyte transformation (LT) and inhibition of leukocyte migration (ILM) from microcapillary tubes or in agarose gel. LT was shown to be the best in vitro correlate of specific lymphocyte sensitization with all antigens. In the ILM assays, PPD showed good correlation with in vivo DCH and in vitro LT; SK-SD showed partial correlation; DO showed no correlation, not being active in any of the ILM tests. Cell distribution and morphology of stained migration patterns, ILM tests performed on separated populations of lymphocytes and polymorphonuclear leukocytes (PMN), as well as the ability of test antigens to stimulate PMN cells to reduce nitroblue-tetrazolium dye, indicated that in ILM tests mononuclear cells were not inhibited in their migration, whereas migration of PMN cells appeared to depend on their direct reaction with the test antigens.     

The Immunomodulatory Effect of Yeast Glucan on Delayed Hypersensitivity

Abstract

The effect of yeast β-1,3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied. Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased. However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR. Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC. Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned.