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.     

The structural basis of cell-mediated immunological reactions of collagen: Characteristics of cutaneous delayed hypersensitivity reactions in specifically sensitized guinea-pigs

Cell-mediated immunological properties of collagen were studied in guinea-pigs employing cutaneous delayed hypersensitivity reactions. The animals were sensitized by a single injection of highly purified native collagen in Freund's complete adjuvant (FCA). Reactivity could be induced with 150 μg of calf collagen. Maximal reactivity was obtained 20 days after sensitization and persisted for more than 3 months. Histologically, the reactions displayed the typical features of delayed reactions with infiltration of predominantly mononuclear cells. No reactivity was induced in animals injected with FCA alone, with collagen in Freund's incomplete adjuvant (FIA), or with guinea-pig collagen in Freund's complete adjuvant. Reactivity was impaired when carrageenan was injected intraperitoneally at the time of challenge. Cyanogen bromide digested collagen was still reactive in sensitization as well as in elicitation. The reaction was found to be species specific in the sense that maximal reactions were obtained when the challenging collagen was from the same species as the sensitizing preparation. In vitro, denatured rat collagen was found to inhibit the migration of macrophages from specifically sensitized animals. By alterations of the immunization schedule antibodies, reactive with collagen in a haemagglutination system, could be induced.

The system lends itself to a comparative investigation of the structural requirements on a natural protein for the induction of the cell-mediated and the humoral immune 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.     

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.     

Studies on cutaneous allergy: Cytophilic antibodies, passive cutaneous anaphylaxis and delayed type allergy

In guinea-pigs a delayed type allergy against sheep erythrocytes (SE) or ovalbumin without detectable circulating and macrophage-cytophilic antibodies was produced by immunization with antigen—antibody complexes. In these animals skin reactions to SE were not enhanced by admixture of macrophages, neither when the macrophages had been sensitized with cytophilic antibodies in vitro, nor when the SE had been (partially) ingested within the macrophages after incubation with opsonizing antibody in vitro.

Also in these animals no enhanced delayed skin reactions to SE were obtained if before the injection of the antigen the skin had been infiltrated with cytophilic antibody or passive cutaneous anaphylaxis (PCA) antibody.

Thus, no enhancing effect of cytophilic antibody on delayed cutaneous hypersensitivity reactions could be obtained.

Furthermore, in guinea-pigs with delayed type allergy to ovalbumin, no enhancing effect of delayed allergy on passive cutaneous anaphylaxis was observed.

     

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.

     

Extracutaneous delayed hypersensitivity, particularly in the guinea-pig bladder

In outbred guinea-pigs with a low level of hypersensitivity to hen egg albumin (HEA) at a time when circulating antibody was not detectable, unaggregated HEA in extracutaneous tissues was rapidly lost from the injection site and did not elicit lesions of delayed hypersensitivity. Aggregated HEA, however, was retained at the depot site and produced lesions in the bladders of similarly prepared guinea-pigs. In Hartley guinea-pigs with a high level of hypersensitivity, as measured by skin reactions, unaggregated HEA, though rapidly dissipated from depot sites, elicited lesions in the bladder. Aggregated HEA, though retained at injection sites, did not produced reactions of delayed hypersensitivity in kidney, testis and muscle. The inflammation of delayed hypersensitivity did not influence the disappearance rate of labelled HEA from the lesion, while the inflammation of an Arthus response was associated with retention of labelled antigen at the injection site.     

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.     

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.

     

Desensitization in vitro—The specific inhibition, by antigen, of the passive transfer of delayed hypersensitivity by peritoneal exudate cells

A preliminary experiment showed that the injection of bovine γ-globulin into guinea-pigs with delayed hypersensitivity to bovine γ-globulin reduced the 24-hour skin reactions to bovine γ-globulin and (to a lesser extent) PPD. The peritoneal exudate cells from the desensitized donors had a reduced ability to transfer delayed hypersensitivity to bovine γ-globulin but a normal ability to transfer delayed hypersensitivity to PPD.

Likewise, it was possible to diminish the passive transfer of delayed hypersensitivity to bovine γ-globulin by peritoneal exudate cells, by exposure of the cells to bovine γ-globulin in vitro. The recipients were tested immediately after cell transfer. This in vitro desensitization was specific, in that the transfer of delayed hypersensitivity to PPD was unaffected.

Exposure of cells in vitro to hypotonic conditions and antibody to guinea-pig γ-globulin did not prevent the passive transfer of delayed hypersensitivity.

     

Cell-mediated and humoral immunity to chick type II collagen and its cyanogen bromide peptides in guinea-pigs.

Cell-mediated immunity (CMI) to chick type II collagen and its cyanogen bromide (CB) peptides was studied in guinea-pigs using cutaneous delayed hypersensitivity reactions. Responses were largely independent of molecular conformation in animals immunized with either native or denatured collagen, and reactions obtained with CB peptides 8, 9, 10 and 12 suggested that sites in the central regions of collagen chains were recognized in CMI. Antibodies to collagen were detected by haemagglutination and immunofluorescence only in animals immunized with native molecule and not in animals immunized with denatured or CB-digested material. Humoral and CMI responses were similar in that neither recognized the pepsin-labile non-helical regions of the molecule. The responses differed in that humoral reactions were conformation-dependent and type-specific and CMI reactions were not.     

The structural basis of cell-mediated immunological reactions of collagen: Reactivity of separated α-chains of calf and rat collagen in cutaneous delayed hypersensitivity reactions

Denatured calf and rat skin collagen and α1- and α2-chains from these collagens were tested for their capacity to sensitize guinea-pigs for the cutaneous delayed hypersensitivity reaction and to elicit this reaction in sensitized animals. Alpha-chains from neutral salt extracted and from urea extracted collagen were also compared. All preparations were fully active as sensitizers as well as elicitors of the skin reaction. In spite of extensive cross-reactions, significant differences between α1- and α2-chains of a given species as well as between corresponding chains of different species could be detected. Differences between chains from neutral salt extracted collagen and from urea extracted collagen were also revealed.     

The antigenicity of native and tyrosylated neutral-salt-soluble rat collagen

Neutral-salt-soluble collagen was extracted from rat skin and purified by repeated precipitation, resolution and dialysis. Part was reacted with N-carboxytyrosine anhydride and a collagen derivative containing 2·6 per cent tyrosine was recovered. Enrichment with tyrosine did not alter the optical rotation, denaturation temperature or electrophoretic mobility of the collagen.

The antigenic properties of native and tyrosylated rat collagen were studied in rabbits and guinea-pigs by micro-complement fixation, tanned cell agglutination and agglutination-inhibition, passive cutaneous anaphylaxis and immediate and delayed skin hypersensitivity. The antigenicity of native collagen was demonstrated. Enrichment with a limited amount of tyrosine enhanced its antigenicity without altering its antigenic specificity and permitted a detailed analysis of the overall specificity of the immunological reaction. Use of the products of controlled degradation of collagen in the immunoassay systems implicitly defined the collagen molecule as responsible for the immune reaction. Collagenase-digestion products still possessed antigenic capacity.

     

In vivo macrophage suppression of delayed hypersensitivity in the guinea-pig.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.     

The antigenic properties of some synthetic polyiminoacids: II. The antigenicity of polypeptides related to collagen; peptides containing hydroxyproline and acetyl-hydroxyproline

The antigenic properties of some synthetic polymers containing hydroxyproline and acetyl-hydroxyproline have been tested in guinea-pigs and rabbits by active cutaneous anaphylaxis, delayed skin hypersensitivity reactions, PCA, tanned cell agglutination and fluorescent antibody microscopy.

The antigenic relationships between these polymers, collagen and acetylated collagen have been investigated. The results obtained suggest that acetyl-hydroxyproline is a common antigenic determinant in both acetylated copolymers and acetylated collagen.

Poly-hydroxyproline and poly-acetyl-hydroxyproline were found not to be antigenic in rabbits or guinea-pigs. Rabbit antiserum against acetylated collagen has been used to stain acetylated tissue sections by immunofluorescence. Absorption studies indicate that acetyl-hydroxyproline groups are important antigenic determinants as shown by the considerable decrease in specific fluorescence when the rabbit anti-acetylated collagen is absorbed with a synthetic polymer containing acetyl-hydroxyproline.

The overall results are discussed in terms of the structural similarity existing between collagen and some of the polymers used in this work.

     

Reversal of antigen-induced resistance to collagen arthritis by cyclophosphamide

Treatment of rats with intravenous injection of 1 mg of soluble native type II collagen induced resistance against the subsequent induction of active arthritis by type II collagen immunization. This resistant state was accompanied by suppressed antibody response and delayed-type hypersensitivity (DTH) skin reaction to type II collagen. However, pretreatment of rats with 20 mg/kg of cyclophosphamide (CY), an agent reputed to damage suppressor T-cell function, 2 days before intravenous injection of soluble type II collagen abrogated the antigen-induced resistance against the subsequent induction of active arthritis. The DTH skin reaction to type II collagen was completely restored and the antibody response to type II collagen was significantly though not completely restored by CY pretreatment. These results provide evidence that antigen-induced resistance to collagen arthritis is mediated, at least in part, under the control of CY-sensitive events.     

Antibodies in delayed hypersensitive rats specifically desensitized by an intravenous injection of various antigen doses

Rats with delayed hypersensitivity to bovine serum albumin (BSA) and diphtheria toxoid were specifically desensitized by an intravenous injection of BSA. The degree of desensitization was positively related to the dose of antigen. The 4-hour skin reactions to BSA were weaker, and the passive haemagglutinin titres, antigen-binding capacity and average avidity of the antibodies were lower in the desensitized rats than in controls. The suppression of delayed hypersensitivity was apparently not caused by increased circulating antibody activity, but by a cellular defect or a humoral blocking factor.     

Selective and specific inhibition of 24 hour skin reactions in the guinea-pig. I. Immune deviation: description of the phenomenon and the effect of splenectomy

Strong 24 hour skin reactions occur in guinea-pigs immunized with antigen in Freund''s complete adjuvant. These reactions were reduced by the injection of 1 mg of the same antigen, in either the soluble or alum precipitated form, 14 days before immunization with antigen in Freund''s complete adjuvant. There was also a reduction in the corneal reaction, which has been regarded as an index of delayed hypersensitivity. This phenomenon was called immune deviation.The phenomenon was demonstrated for bovine γ-globulin, human serum albumin, bovine serum albumin, egg albumin, diptheria toxoid, haemocyanin, purified protein derivative (PPD) and dinitrophenylated proteins. Ten mg of soluble bovine γ-globulin caused considerable reduction of the circulating antibody level and of the 24 hour skin reaction. Alum precipitated bovine γ-globulin and smaller doses of soluble antigen reduced the skin reaction but had less effect on the antibody level. Similar results were obtained with human serum albumin. This suggested that the conditions for eliciting immune paralysis and immune deviation were different.Immune deviation was obtained with either footpad or intravenous injections and with as little as 100 μg of antigen. Alum precipitated bovine γ-globulin caused immune deviation when given 14, 7 or 1 day before or 1 day after immunization with antigen in Freund''s complete adjuvant. It was inactive when given 6 days after immunization. Splenectomy had no effect on the production or deviation of 24 hour skin reactions.Alum precipitated antigen had a variable and usually slight effect on the level of antibody following immunization with the same antigen in Freund''s complete adjuvant. There was, however, a qualitative alteration in the antibody. The sera of guinea-pigs, which had been deviated by a prior injection of bovine serum albumin or bovine γ-globulin, showed only a γ₁ line of antibody on immunoelectrophoresis, while the sera of control guinea-pigs also showed the γ₂ line characteristically seen in guinea-pigs immunized with antigen in Freund''s complete adjuvant.It was concluded that immune deviation was distinct from classical immune paralysis and that the immunologically specific reduction of the 24 hour skin reactions might be due, at least in part, to a selective loss of delayed hypersensitivity.     

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.

     

The immunological significance in the guinea-pig of in vitro transformation of lymph node, spleen and peripheral blood lymphocytes

Cells from lymph nodes or spleen, or peripheral blood leucocytes of immunized guinea-pigs were cultured in the presence of antigens or phytohaemagglutinin. Significant incorporation of tritiated thymidine occurred in a variable proportion of the experiments with lymphocytes from each of the three sources. Cells taken from animals that had been immunized with sheep erythrocytes with adjuvant, and which showed strong delayed hypersensitivity, and from animals immunized intravenously with sheep erythrocytes, which failed to show delayed hypersensitivity reactions, both responded to sheep erythrocytes in vitro.

Cells from guinea-pigs immunized with complete Freund's adjuvant alone, which showed strong delayed hypersensitivity to tuberculin PPD, gave more positive responses in vitro than did cells taken from animals which received an intravenous injection of tuberculin PPD before the adjuvant. These animals showed no or weak delayed hypersensitivity reactions (immune deviation).

The immunological significance of the in vitro proliferative reaction is discussed.