Modification of delayed-type hypersensitivity reactions to ovalbumin in cyclosporin A-treated guinea-pigs.

Cyclosporin A (Cs A) administered daily (25 mg/kg per os) to outbred guinea-pigs for 2 weeks following immunization with ovalbumin (OVA; CsA 0-13) caused profound suppression of 14-day delayed-type hypersensitivity (DTH) skin reactions. Very marked impairment of DTH was also found when Cs A was given for the first time 24 hr before skin testing and at 6 and 24 hr thereafter. In contrast, Cs A given on days 0-4 following OVA immunization (Cs A 0-4) caused dose-related potentiation of 14-day skin responses. These changes in the magnitude and character of DTH in vivo were accompanied by striking alterations in lymphocyte transformation responses and in the extent of macrophage migration inhibition and lymphokine production. Whereas Cs A (0-13) caused almost total suppression of the mitogenic responses of lymph node cells to PHA and antigen, OVA-induced migration inhibition and production of the lymphokine inducing macrophage procoagulant activity (MPCA), Cs A (0-4) augmented these responses to OVA, but did not affect lymphocyte transformation or lymphokine production in response to mitogen. Strain 13 guinea-pigs treated with Cs A (0-4) showed depressed Arthus, but augmented DTH responses to OVA. This significant increase in cell-mediated immunity could be passively transferred using spleen and peritoneal exudate cells, suggesting that under these circumstances Cs A (0-4) may interfere with the generation of a population of suppressor cells which regulate DTH reactions in the guinea-pig.     

Immunoregulatory effect of antibody on delayed-type hypersensitivity in mice

We analyzed the conditions for in vivo toleration of delayed-type hypersensitivity (DTH). The intravenous injection of a high dose of keyhole-limpet hemocyanin (KLH) into BALB/c mice did not induce DTH in vivo, but the serum titers of the anti-KLH antibody were significantly elevated. This lack of DTH response was antigen-specific, and the intravenous injection of the antigen induced effector-phase suppressor T cells. The findings suggested that the lack of a DTH response brought about by the intravenous injection of a high dose of antigen was not immunological tolerance. Treatment with a high dose (250 mg/kg) of cyclophosphamide - but not a low dose (50 mg/kg) - enhanced the DTH, but suppressed antibody production. These results indicate that humoral immune response participate in the regulation of DTH. In addition, the transfer of serum or immunoglobulin from mice that were injected intravenously with a high dose of the antigen suppressed the DTH. We concluded that the antibodies regulate DTH in the antigen-specific manner.     

IgG antibody and delayed-type hypersensitivity responses in nude mice grafted with thymic epithelium.

This study compared the ability of foetal thymic epithelium depleted of lymphocytes and dendritic cells, by low temperature or deoxyguanosine (dGuo) treatment in organ culture, to reconstitute T-cell function in nude mice. It is shown that renal capsule grafts of either type could promote the development of functional T lymphocytes in the periphery, as judged by in vivo assays. Both syngeneic and allogeneic thymic epithelium endowed nude mice with the capacity to mount IgG antibody and delayed-type hypersensitivity (DTH) responses to the T-dependent antigen ovalbumin (OVA). Functional reconstitution was accompanied by the appearance of Thy-1-bearing cells in the spleens of thymic grafted nude mice. The results from allogeneically grafted recipients show that a substantial population of peripheral T cells was present that collaborated with B cells and other antigen-presenting cells (APC) which do not express major histocompatibility complex (MHC) molecules of the thymus donor haplotype.     

Immune complex-mediated enhancement of antibody responses without induction of delayed-type hypersensitivity

Immunoglobulin (Ig)G and IgE antibodies enhance the humoral response in vivo to soluble antigens with which they form complexes. In vitro, antigen is targeted to B cells by IgE antibodies and to macrophages and dendritic cells (DCs) by IgG, thus leading to increased antigen presentation to specific T cells. Possibly these mechanisms are also responsible for antibody-mediated enhancement in vivo. We now address the question of whether IgG- and/or IgE-antigen complexes can prime for delayed-type hypersensitivity (DTH), a reaction known to require primed T helper (Th)1 cells. Mice were immunized with IgG-anti-2,4,6-trinitrophenyl (TNP)/BSA-TNP or IgE-anti-TNP/BSA-TNP. Mice given BSA-TNP alone or BSA-TNP in complete Freund's adjuvans (CFA) were used as controls. DTH and IgG-anti-BSA levels were measured after subsequent challenge with BSA. A potent BSA-specific antibody response was induced by IgE- or IgG-complexed antigen as well as by CFA/antigen but DTH-reactions were only observed in mice immunized with CFA/antigen. Both IgE and IgG enhanced the production of BSA-specific IgG1, IgG2a and IgG2b, although the most pronounced enhancement was seen in the production of IgG1. These findings suggest that Th2 cells rather than Th1 cells are involved in the immune response to IgG- and IgE-immune complexes.     

The effects of anticoagulants and other drugs on cellular and cutaneous reactions to antigen in guinea-pigs with delayed-type hypersensitivity

Delayed-type hypersensitivity to tuberculin was induced in guinea-pigs by vaccination with BCG. The effects of several drugs on the responses of peritoneal exudate cells to tuberculin (PPD) and on delayed skin reactions to PPD were investigated. In untreated animals intraperitoneal injections of PPD were followed by the virtually complete loss of macrophages from the exudates (the macrophage disappearance reaction), the partial loss of lymphocytes and a marked increase in the number of polymorphs in the exudates. The macrophage disappearance reaction was markedly or completely inhibited in animals treated with the anticoagulant drugs heparin or sodium warfarin, very slightly inhibited in animals treated with cortisone acetate or promethazine and not inhibited in animals treated with reserpine. The other peritoneal cellular responses were variably but only slightly affected by these drugs. Delayed skin reactions to PPD were partly inhibited in animals treated with heparin, sodium warfarin or cortisone acetate and more strongly inhibited in animals treated with a combination of sodium warfarin and cortisone acetate. Histological examination of the skin test sites of untreated animals and of animals treated with sodium warfarin and/or cortisone acetate showed that the accumulation of macrophages was more markedly inhibited in animals treated with sodium warfarin than in animals treated with cortisone alone. No correlation could be established between the effect of treatment with sodium warfarin on the macrophage disappearance reaction, on blood coagulation and on serum complement levels.     

Circulatory basophilia in guinea pigs with delayed-type hypersensitivity

Circulatory basophilia could be induced in inbred guinea pigs systematically immunized with ovalbumin and consequently provoked repeatedly with dissolved ovalbumin applied onto the mucosa of the nares or the outer eye. The degree of the increase in circulatory basophil granulocytes depended on the adjuvant used and was significantly more pronounced after immunization with Freund's complete adjuvant than with alhydrogel (A1(OH)3). The degree of basophilia was also dependent on the animal strain, but different in two strains selected for high-asthma trait.     

Cytophilic activity of IgG2 from sera of guinea-pigs immunized with bovine gamma-globulin

IgG2 from immune guinea-pig sera collected 1-2 weeks after secondary BGG challenge showed considerably enhanced cytophilic activity; the preparations contained aggregates (±10 per cent of the total immunoglobulin) shown by antigen exchange to be immune complexes with a probable composition Ag (fragments) Ab₅. After removal of aggregates the cytophilic activity of immune 7S IgG2 approximated that of IgG2 from unimmunized animals.Artificial immune complexes from either non-immune or immune 7S IgG2 (aggregated with the F(ab'')₂ fragments of rabbit anti-guinea-pig Fab) showed greatly enhanced cytophilic activity, maximal at antigen: antibody equivalence and comparable for both IgG2 preparations.These data are consistent with the view that cytophilic activity for macrophages is a uniform property of the whole guinea-pig IgG2 class.The IgG2 complexes apparently bind to glass-adherent cells and also to a population of non-glass-adherent mononuclear cells, present in peritoneal exudates but not detectable in peripheral blood.     

Cytophilic activity of IgG2 from sera of unimmunized guinea-pigs

The interaction between 7S IgG2 from unimmunized guinea-pigs and macrophage-rich peritoneal exudate cells from oil-stimulated guinea-pigs was examined by ¹²⁵I-labelled IgG2 uptake.

The cell uptake of ¹²⁵I-labelled IgG2 reaches a maximum after 90 minutes at 20°; increased levels of labelling (1 to 23 atoms of iodine per immunoglobulin molecule) lead to slightly enhanced uptake, but the effect does not exceed experimental variability.

Peripheral white blood cells bind relatively low levels of IgG2; the uptake by peritoneal cells is attributable primarily to a subpopulation of glass-adherent cells, presumably macrophages.

Binding of IgG2 to macrophages is reversible even with a mild washing procedure. Repeated washing of cells did not affect the association constant, but the apparent number of receptor sites per cell was progressively reduced. After correction for the washing procedure employed, the number of IgG2 receptor sites was estimated as 2.5±0.4 x 10⁶ per cell; the association constant (Ka) was 1.46±0.45 x 10⁶ L/M at 20°.

     

Increased delayed-type hypersensitivity reaction in rats neuromanipulated with capsaicin

We studied the neuroimmunomodulatory effect of capsaicin on the development of delayed-type hypersensitivity (DTH) reaction and antibody formation after reduction of neuropeptides in the peripheral sensoric nervous system. Rats were sensitized with picryl chloride on their shaved abdomen or by subcutaneous injections with picrylsulfonic acid (PSA). Manipulation of the content of neuropeptides in the sensory nerve system with capsaicin was performed 1 week before or after sensitization. An increase of the DTH reaction assessed as increased ear thickness after challenge was seen especially when the rats were given capsaicin after sensitization. The formation of specific antibodies was not affected, although the level of total IgG was decreased in PSA-sensitized rats treated with capsaicin after sensitization. The results demonstrated that the DTH reaction in rat was slightly but significantly affected by the abolishment of neuropeptides in sensoric nerve endings with capsaicin. Even if the direct effect of capsaicin on the immune system still has to be elucidated, these results indicate that neuropeptides in sensory nerves are involved in the pathogenesis of DTH.     

Induction of delayed-type hypersensitivity with BCG-associated proteins.

Microgram quantities of bovine serum albumin (BSA) and hen egg albumin (OA) associate spontaneously with living BCG in aqueous suspension. The association is stable insofar as bound protein cannot be readily dissociated from the organism by repeated washing. Association of BSA or OA is dependent upon the concentration of specific protein or competing protein in the incubation mixture, pH and the amount of protein already bound to the organisms. Washed BCG "complexes" containing either BSA or OA are potent inducers of delayed-type hypersensitivity (DTH). The same soluble proteins are far less effective inducers of DTH, even when injected with BCG. The capacity of OA-BCG complexes to provoke a cell-mediated response seems to be related, at least in part, to "stabilization" of the antigen on an appropriate carrier and its concentration at the site of BCG injection.     

P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity

The role of P-selectin in T lymphocyte accumulation and injury was studied in delayed-type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti-rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte-dependent macrophage influx and injury characteristic of DTH. Up-regulation of P-selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti-CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up-regulation endothelial cell P-selectin. Treatment with anti-CD4 antibody produced similar results. Blocking P-selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up-regulation of endothelial cell P-selectin was prevented. These data demonstrate that P-selectin is locally up-regulated on endothelial cells in T cell-dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P-selectin in local T cell recruitment and subsequent injury in DTH.     

Impaired delayed hypersensitivity in germ-free guinea-pigs

The delayed-type hypersensitivity response of germ-free guinea-pigs was found to be defective. Whereas almost all conventionally reared guinea-pigs became hypersensitive to an allergenic hapten (picryl chloride), most germ-free guinea-pigs did not. When injected with a fully antigenic substance, bovine γ-globulin (BGG), none of the germ-free animals acquired BGG-specific delayed hypersensitivity. Further, none of the germ-free guinea-pigs developed spontaneous iso-hypersensitivity for a beta globulin as do conventional guinea-pigs. In addition, germ-free guinea-pigs given Freund's complete adjuvant did not develop the characteristic induration or erythema normally seen at injection sites and most animals died within 21 days.

Germ-free guinea-pigs given competent lymphoid cells from highly sensitized conventional guinea-pigs were unable to translate adoptive hypersensitivity into delayed dermal reactions.

A permeability factor in aged guinea-pig sera, injected into the skin of germ-free and conventional animals to determine whether the skin of germ-free guinea-pigs was able to support reactions, initiated immediate dermal reactions of equal intensity in both sets of animals.

     

Cross-reactivity between human adenoviruses in delayed-type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed-type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient-purified human Ad serotype 6 (Ad6) induced DTH in a dose-dependent manner. In Ad6-sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross-reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross-reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.     

Regulation of delayed-type hypersensitivity V. Suppressor cell memory in antigen-specific suppression of delayed-type hypersensitivity to sheep erythroeytes

Mice primed i. v. with 10⁹ sheep red blood cells (SRBC) produce antigen-specific T suppressor (Ts) cells which inhibit both the induction and the expression of delayed-type hypersensitivity (DTH). These Ts cells are detectable in the spleen and lymph nodes 3–5 days after priming but are largely absent by 6 days. The transient detect-ability of the Ts cells contrasts sharply with the profound antigen-specific suppression which persists in primed donor mice for at least a year. Evidence is presented that this long-term impairment of DTH is maintained, at least in part, by memory Ts cells which are Thy-1⁺, cyclophosphamide-resistant and antigen-specific. Although they appear to be co-induced with the short-lived primary Ts cells and localize initially in the lymphoid organs, they are present in the long-lived circulating pool of T cells and can be adoptively transferred by celomic parabiosis. Memory Ts cells are readily reactivated by lower doses of SRBC which would induce T effector cells rather than Ts cells in naive animals. Reactivated memory Ts cells seem to generate a population of antigen-specific secondary Ts cells which again localizes in the lymphoid organs and can adoptively suppress the induction and expression of DTH to SRBC.     

Increased hyaluronic acid is associated with dermal delayed-type hypersensitivity

Rabbits sensitized subcutaneously with heat-killed bacilli Calmette-Guerin (BCG) and challenged intradermally with heat-killed BCG or purified protein derivative (PPD) demonstrated classical dermal delayed-type hypersensitivity reactions which peaked two days postchallenge. Animals challenged with BCG developed dermal granulomas as measured by induration and gross observation. Challenge with either PPD or BCG resulted in increased levels of dermal hyaluronic acid (HA) by two days postchallenge. Dermal HA returned to normal levels by seven days postchallenge regardless of the challenge antigen. These results indicated that increased HA is associated with dermal delayed-type sensitivity, but increased HA is not associated with dermal granulomatous hypersensitivity. These results are in contrast to previously reported work which indicates that increased HA is associated with both pulmonary delayed hypersensitivity and pulmonary granulomatous hypersensitivity.