The structural basis of cell-mediated immunological reactions of collagen: The species specificity of the cutaneous delayed hypersensitivity reaction

Guinea-pigs were sensitized for delayed hypersensitivity skin reactions with native rat, rabbit, fish, or human collagen or with (L-Pro-Gly-L-Pro)_n, mixed with Freund's complete adjuvant (FCA). Mammalian collagens were more immunogenic than fish collagen and the synthetic, collagen-like polymer. The terminal, non-helical portions of calf and rat collagen were not required for expression of species specific cell-mediated reactions of these collagens. Although the animals could readily distinguish between mammalian collagens on one hand and fish collagen and (L-Pro-Gly-L-Pro)_n on the other, the discriminatory capacity was best expressed among each of the two groups of antigens.     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. II. Mechanism of the phenomenon

Recipient guinea-pigs pretreated with Freund's complete adjuvant alone show depressed 4 hr (Arthus) reactions following passive transfer of antiserum to bovine γ-globulin and human serum albumin. Recipient outbred guinea-pigs and inbred rats also show depressed 24 hr delayed reactions following passive transfer of immune peritoneal exudate cells and serum. This indicates that Freund's complete adjuvant depresses certain inflammatory responses.

Donor guinea-pigs and inbred rats pretreated with FCA alone and then immunized with BGG in FCA transfer smaller 24 hr reactions to normal recipients than comparable donor animals which have not been pretreated with FCA. This suggests that pretreatment with FCA depresses the central state of delayed hypersensitivity which normally follows immunization with BGG in FCA.

These two findings may explain why pretreatment with FCA depresses the 4 and 24 hr skin reactions which otherwise follow immunization with antigen in FCA.

     

Specific suppression of delayed hypersensitivity skin reactions to collagen in guinea-pigs after immunization with collagen and Freund''s incomplete adjuvant.

Partial suppression of cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs was induced by pre-immunization with collagen and FIA. This suppression is specific since: (a) pretreatment with OA and FIA or FIA alone did not cause suppression of skin reactions to collagen; (b) suppression was observed only if the collagen used for pretreatment was from the same species as that employed for sensitization for delayed hypersensitivity reactions; and (c) animals with depressed skin reactivity to collagen reacted normally to PPD. The suppression is not mediated by inducible, circulating antibodies to collagen since: (a) antibody titres measured by passive haemagglutination did not correlate with the degree of suppression; (b) suppression was observed with collagen in random coil conformation which sensitizes guinea-pigs for delayed hypersensitivity skin reaction but does not induce antibodies to denatured collagen; (c) best suppression was obtained if the animals were pretreated with collagen and FIA 3 days before the sensitizing injection; and (d) passively transferred antibody from animals with suppressed skin reactivity did not suppress skin reactivity of animals made hypersensitive to collagen by injection of collagen and FCA.     

Temporal relationship between tuberculin skin reactivity and in vitro mitotic response

1. Albino guinea-pigs were actively sensitized with complete Freund's adjuvant.

2. Tuberculin skin reactivity and tuberculin-induced in vitro mitotic responsiveness of circulating lymphocytes were determined sequentially in each animal after sensitization.

3. In nine of ten animals studied, both skin reactivity and in vitro mitotic response to tuberculin appeared 3 weeks after sensitization.

     

Immunological responses following injection of antigens in Freund's adjuvant into thymus and other tissues

Groups of young adult rats were inoculated with bovine serum albumin in complete Freund's adjuvant into one foot-pad, a cervical lymph node, the spleen, a forelimb muscle, the cavity of the mediastinum or directly into the thymus. Arthus responses and circulating antibodies occurred in decreasing intensity in the order listed, but marked delayed sensitization resulted in the foot-pad and lymph node groups only.

Groups of guinea-pigs were inoculated with egg albumin in complete Freund's adjuvant into a foot-pad, on the surface of the thymus or into the thymus. Delayed sensitization did not develop until the 17th day in the intrathymic group, and Arthus reactions and circulating antibodies were poor when compared with the other animals.

Allergic encephalomyelitis was found in all seven rats injected with spinal cord and adjuvant in one foot-pad and in five of seven animals injected into a cervical node, while a single minimal lesion was present in one of twelve animals inoculated into the thymus directly.

Plasma cells were seen in the thymus after injection of protein and adjuvant near the connective tissue of the capsule, the interlobular septa and blood vessels, whereas nervous tissue and adjuvant produced virtually no plasma-cell response.

     

Dependence of the antibody response of guinea-pigs to collagen on the type of adjuvant and on the conformation of the antigen.

The immunological response to collagen of guinea-pigs is strongly dependent on the conformation of the antigen and on the type of adjuvant. Freund's complete adjuvant facilitated excellent delayed hypersensitivity skin reactions to native (triple helical conformation) as well as denatured (random coil conformation) collagen. Immunization of guinea-pigs with collagen in this adjuvant gave rise to very low levels of antibody to native collagen and failed to induce antibodies to denatured collagen. Use of Freund's incomplete adjuvant resulted in excellent antibody responses to native collagen, but it did not induce antibodies to denatured collagen. Animals injected with collagen and Freund's incomplete adjuvant were not sensitized for cell-mediated immunological reactions. The antibodies to collagen were specific with regard to collagen from various species but displayed different degrees of cross-reactivities depending on the species of collagen used for immunization. They were specific for the triple helical conformation of the collagen molecule.     

Transfer of delayed Hypersensitivity by Lymph-Node Cells in Testis Autosensitization

Guinea pigs sensitized by an extract of homologous testis and Freund's adjuvant developed delayed skin hypersensitivity towards a purified testis antigen. When lymph-node cells from sensitized animals were transferred into normal guinea pigs by intravenous, intraperitoneal or intracutaneous injection the recipients also developed delayed skin hypersensitivity.

Maximum reactions in recipients were obtainable after the transfer of cells from donors which had been sensitized for only 6 days. The recipients became sensitized immediately after cell transfer but their sensitization lasted only a few days.

It can be concluded from this and earlier work that sensitized cells as well as free circulating antibody play a part in testis autosensitization.

     

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.

     

Studies on delayed hypersensitivity to hepatitis b antigen in chimpanzees

Twenty-eight chimpanzees were divided into six groups according to their history of previous immunization or exposure to hepatitis B antigen (HBAg) and studied for delayed hypersensitivity (DH) to HBAg. Purified HBAg derived from a normal human carrier was used for in vivo skin testing and in vitro leucocyte migration inhibition tests. Of seventeen chimpanzees immunized with HBAg in Freund's complete adjuvant (FCA), nine exhibited positive DH reactions to HBAg with good correlation between the in vivo and in vitro responses. Of the seventeen chimpanzees, fourteen also exhibited positive DH reactions to purified protein derivative of tuberculin (PPD) with marked erythema and induration; the other three exhibited only erythema with no induration. None of the seventeen animals exhibited any immediate reactivity to either HBAg or PPD. Intradermal injection of HBAg-negative human serum failed to elicit DH reactions in four animals who showed positive skin test with purified HBAg; the DH response was thus probably HBAg-specific. Nineteen chimpanzees, including six unimmunized animals, three chronic carriers of HBAg and two which had been injected with HBAg without FCA, failed to show DH response to HBAg. Thus, DH to HBAg was observed only in animals hyperimmunized with HBAg in FCA.     

The structural basis of cell-mediated immunological reactions of collagen: Recognition by the cutaneous delayed hypersensitivity reaction in guinea-pigs of conformational alternations of rat and calf skin collagen

Guinea-pigs were sensitized for delayed cutaneous hypersensitivity reactions to native and thermally denatured rat and calf skin collagens. Native and denatured collagens sensitized the animals to a comparable degree. However, the immune mechanism of the animals differentiated the purely conformational alterations of the molecule, even across the species barrier. Denaturation was accompanied by loss of immunogenic information as well as by exposure of new determinants, not recognizable in the native molecule. The sera of these animals were examined for the presence of antibodies reactive with native collagen in a passive haemagglutination system. Low levels of such antibodies were found in animals sensitized with native collagen and no antibodies reactive with native collagen in animals sensitized with denatured molecules although the latter group of guinea-pigs responded well with delayed skin reactions to native collagen.     

Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: evidence for tuberculin type delayed hypersensitivity of the reaction.

Delayed hypersensitivity (DH) reaction can be induced in mice by intravenous sensitization with sheep erythrocytes (SRBC). However, as the sensitizing procedure is quite different from a usual mode of sensitization for DH using complete Freund's adjuvant (FCA), the nature of this reaction has been a matter of controversy. In an attempt to characterize this reaction, we placed special interest on two possibilities regarding the nature of this reaction; Jones-Mote reaction or tuberculin type DH. From the kinetics study on the DH after challenge, the DH reaction to SRBC in mice by intravenous sensitization was clearly distinguished from the Arthus reaction. The dose-response pattern of this reaction also suggested that the contribution of Arthus reactivity to delayed reactivity was negligible. Cell reconstitution experiments revealed this DH to be quantitatively thymus cell dependent. Furthermore, this DH required macrophages at its manifestation stage, and appearance of basophil infiltration at the lesion was absent. In addition, strain difference and ageing of host mice influenced the DH reaction in exactly the same fashion in which these factors influence the tuberculin type-DH induced by subcutaneous sensitization with methylated human serum albumin (MHSA) in FCA. Taken collectively, it was concluded that this DH reaction can be categorized as the tuberculin type.     

Prevention of experimental allergic encephalitis in guinea-pigs with spinal cord protein: optimum pretreatment schedules and appraisal of plausible mechanisms.

Pretreatment of Hartley guinea-pigs with three injections of 100 microgram of the purified bovine spinal cord protein, (SCP), protected tham from clinical experimental allergic encephalitis (EAE) when they were subsequently challenged with 50 microgram of purified bovine myelin basic protein (MyBP) in Freund's complete adjuvant (FCA). The length of the optimum pretreated schedule was found to be 1 week and the animals were fully protected for 2 weeks. Protection declined thereafter so that by 4 weeks following pretreatment only 40% of the animals were protected. The protected state could be restored several weeks after it had lapsed by repeating the treatment with SCP. Immature animals did not respond well to pretreatment with SCP. Evidence was presented to indicate that specific, non-specific immune suppression or anti-SCP IgG were not involved to a significant extent in protection. SCP-treated guinea-pigs regularly displayed delayed skin hypersensitivity to MyBP after sensitization but there was no correlation between the degree of cutaneous reactivity and protection from disease. Because SCP is localized in the nerve axon, the speculation was advanced that anti-SCP blocking factors might be responsible for preserving axonal function in SCP-treated animals sensitized with MyBP.     

The relation between the cell-mediated immunological response and the induction of circulating antibodies to collagen in guinea-pigs.

Cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs were partially but specifically suppressed if the animals had been pretreated with collagen and Freund's incomplete adjuvant. Such animals responded normally to skin-reactive factor prepared with ovalbumin. Lymphoid cells from animals with normal delayed hypersensitivity to collagen functioned normally in animals with suppressed skin reactivity. Cells from animals with suppressed delayed hypersensitivity were specifically, functionally impaired since they transferred delayed hypersensitivity into neutral recipients efficiently for PPD but not for collagen. Suppression could be induced in Cy-treated animals, and it persisted for at least 143 days. It is concluded that guinea-pigs with depressed delayed hypersensitivity to collagen are functionally impaired with respect to those T cells normally generated by induction of delayed hypersensitivity.     

Interaction between `sensitized lymphocytes' and antigen in vitro: IV. Studies on the mechanism of release of skin reactive and macrophage migration-inhibitory factors

Peritoneal exudate lymphocytes (PEL) of guinea-pigs injected with Freund's complete adjuvant (FCA) were capable of liberating skin reactive factor (SRF) and macrophage migration-inhibitory factor (MIF) when cultured for 24 hours in the presence of sheep red cells (SRC) preincubated with lymph node cells (LNC) of guinea-pigs immunized with SRC in FCA. Similar release of soluble mediators was found in the following situations: (a) incubation of non-immune PEL with SRC sensitized with the exclusion peak of Sephadex G-200 fractionated immune LNC supernatant; (b) incubation of non-immune LNC with complexes of SRC and anti-SRC IgG, prepared from serum of animals immunized with SRC in CFA, and (c) incubation of non-immune LNC with a mixture of SRC urea extract and anti-SRC IgG. SRC incubated for 24 hours with immune LNC and freed of lymphocytes by selective filtration through a Millipore filter, were able to provoke an inflammatory reaction in the skin of normal guinea-pigs, which was delayed in character. These findings are interpreted as suggestive evidence for the non-identity of the mediator-producing and antigen-reactive cells and as support for the hypothesis that a complex composed of antigen and an antibody-like material, secreted by stimulated antigen-reactive lymphocytes, is responsible for the release of soluble mediators in cell-mediated immune reactions.     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Lymph node localization and whole body distribution of radioiodinated encephalitogenic polypeptide in guinea-pigs

A bovine encephalitogenic polypeptide (BEP) labelled with radioiodide retained its capacity to induce experimental encephalomyelitis (EAE). Guinea-pigs were injected with ¹²⁵I BEP in Freund's complete adjuvant (FCA), to study changes in the architecture and the distribution of radioactivity in draining lymph nodes, and the amount of radioactivity in various organs.

After injection of BEP in FCA the lymph node rapidly enlarged. Within 48 hr there was depletion of lymphocytes, the enlarging lymphoid follicles had become confluent and there was proliferation of large `epithelioid' cells throughout the node. At 5 days the lymph node architecture was disorganized and lymph follicles with germinal centres could not be recognized; similar but less pronounced changes were present in regional nodes. By contrast, after injection of flagellin in FCA, there were numerous lymphocytes, plasmablasts and pyroninophilic cells, germinal centres were prominent, and the architecture was preserved.

From 0·5 to 0·8% of the total injected radioactivity was concentrated in the popliteal lymph node 2–5 days after injection of ¹²⁵I BEP in FCA. No radioactivity was concentrated in the node after injection of ¹²⁵I BEP without FCA, and animals thus immunized did not develop encephalomyelitis.

The popliteal lymph node was examined by autoradiography after injection of ¹²⁵I BEP in FCA. At 24 hr radioactive encephalitogen associated with droplets of adjuvant was present mainly in the peripheral sinus and at 48 hr encephalitogen–adjuvant droplets were deposited randomly throughout cortex and medulla. These droplets appeared to represent sites where lymphoid cells acquired their capacity for pathogenic reactivity with their target antigen in the central nervous system.

     

Cytophilic antibody in guinea-pigs with delayed-type hypersensitivity

Cytophilic antibody has been demonstrated in the serum of guinea-pigs injected with sheep red cells mixed with Freund's complete adjuvant. Sheep red cells adhered, in the absence of serum, to normal guinea-pig macrophages which had been treated in vitro with such serum. Treatment of polymorphs and lymphocytes with the antiserum did not confer on them this capacity to adsorb sheep red cells. The animals immunized with sheep red cells and complete adjuvant, when skin tested with an extract of sheep red cells, showed strong delayed-type reactions.

Cytophilic antibody was not detectable in the serum of the majority of guinea-pigs which received sheep red cells mixed with incomplete adjuvant. Skin tests in these animals produced strong Arthus reactions but no delayed reactions. Titres of haemagglutinating antibodies were similar whether the adjuvant was complete or incomplete.

     

Hapten-specific antibodies in allergic contact dermatitis in the guinea pig

A guinea pig model was used to investigate the potential role of hapten-specific antibodies in allergic contact dermatitis. Hapten-specific antibodies of both the IgG1, IgG2 and IgM (sub)classes could be readily detected by ELISA in sera obtained after single or repeated exposure to the allergens 2,4-dinitrochlorobenzene (DNCB) or 4-ethoxymethylene-2-phenyloxazolone. Experiments in which animals were strongly boostered with Freund's complete adjuvant support previous reports that hapten-specific antibodies are able to transfer contact skin reactions with a delayed time course into naive recipients. In contrast, epicutaneous sensitization procedures never allowed transfer of such reactivity. Upon transfer of immune sera to previously sensitized animals, existing allergic contact dermatitis was unaffected, except for a distinct suppression of induration, but not erythema, in the DNCB system. In actively sensitized animals, antibody-related suppression of induration was never observed. Hapten-specific hyposensitivity, as induced by repeated epicutaneous exposure, was also not related to circulating antibody levels. Our findings do not support the view that hapten-specific antibodies should be reconsidered as playing a potentially important role in allergic contact dermatitis.     

Cellular immunity to Legionella pneumophila in guinea pigs assessed by direct and indirect migration inhibition reactions in vitro

Spleen cell cultures from guinea pigs given legionella pneumophila vaccine in complete Freund adjuvant or as a sublethal infection were inhibited in their migration activity in vitro when incubated with specific antigen. Both direct and indirect migration inhibition assays revealed sensitization of the guinea pigs to the bacterium, with demonstrable reactivity 25 to 40 days or more after sensitization. No consistent reactions occurred when the guinea pigs were given the killed Legionella vaccine in incomplete Freund adjuvant in saline. However, spleen cells from guinea pigs injected with sublethal doses of the Legionella vaccine 3 to 4 weeks earlier showed positive migration inhibition factor reactivity. Cutaneous hypersensitivity and lymphocyte blastogenic responsiveness in vitro also developed in guinea pigs sensitized with killed Legionella vaccine in complete adjuvant or given a sublethal infection with the bacterium. These results indicate that in vitro assays for migration inhibitory activity may be utilized to monitor the development of the sensitization of guinea pigs to L. pneumophila, and such reactions correlate with skin reactivity and in vitro lymphocyte blastogenic responses.     

The Prevention of Delayed Hypersensitivity to Homologous Serum and Transplantation Antigens in Guinea Pigs

The injection of normal guinea-pig serum in Freund's complete adjuvant into guinea pigs other than the serum donor led to the development of a long-lasting, delayed hypersensitivity. Serum alone, without adjuvant, had no sensitizing capacity. Circulating antibodies to the allotypic antigens could not be detected.

Injection of as much as 8 ml. of serum into sensitized animals did not achieve desensitization. However, the intravenous injection of the same amount of serum prevented normal guinea pigs from becoming sensitized to the same antigen for over 100 days. This unresponsiveness was interpreted to be due to an interference by the serum with the process of sensitization.

This method of producing unresponsiveness was applied to the homograft reaction: guinea pigs, given a series of intravenous injections of spleen extracts containing transplantation antigens, could not be subsequently sensitized by the injection of spleen cells from the same donors. However, immunization provided by skin grafting could probably break through this unresponsiveness: guinea pigs, judged to be unresponsive by the intradermal injection of spleen cells before skin grafting, all developed an intense cutaneous hypersensitivity after they had rejected the graft.