The Fate of [131I]Labelled Antigens in the Skin of Normal Guinea-Pigs and those with Delayed-Type Hypersensitivity

A comparison was made of the retention of three antigens PPD (protein purified derivative of tuberculin), HSA (human serum albumin) and HGG (human γ globulin) labelled with ¹³¹I in the skin of normal guinea-pigs. It was found that PPD was eliminated more rapidly than HSA or HGG in the first 24 hours after skin test. In contrast PPD was found to be more readily adsorbed to collagen and cellular tissues in vitro.

The retention of these antigens at 24 hours in normal and sensitized guinea-pigs was also compared. It was found that PPD was retained in the tuberculin lesion as compared with normal skin, whereas HSA was eliminated more rapidly from a delayed-type hypersensitivity lesion to HSA when compared with normal skin. It was concluded that retention of PPD was probably not specific to delayed-type hypersensitivity but was related to the difference in character and severity of the inflammatory process, which itself was related to the chemical nature of the antigen involved.

     

The Passive Transfer of Delayed Hypersensitivity in Guinea Pigs by the Transfusion of Isotopically-Labelled Lymphoid Cells

Delayed hypersensitivity to tuberculin and contact sensitivity to picryl chloride were transferred passively to normal guinea pigs by the intravenous injection of spleen, lymph node and peripheral blood mononuclear cells labelled in vitro with ⁵¹Cr or in vivo with ³²P or ³H-thymidine. No significant difference was found between the number of injected cells arriving at the passive lesions and actively induced lesions in controls, after 24 hours. The proportion of labelled cells in the exudate was the same as that in the peripheral blood and they showed a random distribution throughout the lesion.

The fate of labelled cells in the body was followed, especially their role of elimination from the lungs and peripheral blood.

It was concluded that no special affinity of the transferred cells for antigen could be demonstrated after 24 hours.

     

Simultaneous measurements of the accumulation of isotope-labelled protein and erythrocytes in skin reactions of allergic inflammation in the guinea-pig

This paper describes a method for simultaneous measurement of the accumulation of plasma protein and erythrocytes in skin reactions of hypersensitivity to bovine γ-globulin (BGG) and tuberculin PPD in the guinea-pig. The procedure consists in giving ¹²⁵I-labelled plasma albumin and ⁵¹Cr-labelled autologous erythrocytes together by intravenous injection into guinea-pigs bearing skin lesions of allergic inflammation at different times and for different periods during the development of the skin reactions. Isotope accumulation in excised skin reactions is measured by scintillation counting at different stages during the evolution of hypersensitivity responses.

Skin reactions of combined anaphylactic and Arthus hypersensitivity to BGG were characterized by pronounced increased vascular permeability principally in the first hour. In established 24-hour hypersensitivity reactions to both antigens (BGG and PPD) there was continuing accumulation of both plasma albumin and erythrocytes. During the development of the tuberculin reaction, an intermediate phase of isotope accumulation occurred between 6 and 9 hours after skin testing; serum transfer studies showed that this intermediate peak was not attributable to circulating antibody alone. These isotope tracer techniques were also applied to study vascular permeability in systemically transferred reactions of delayed hypersensitivity and in the vascular response to the intradermal injection of an inflammatory factor generated by antigen-activation of sensitized lymphocytes.

It was concluded that isotope tracing provided objective and sensitive methods for analysing microvascular responses in allergic inflammation.

     

Transfer of delayed and Arthus sensitivity with blood plasma from X-irradiated guinea-pigs

Lethal X-irradiation of sensitized guinea-pigs failed to release a mediator of delayed-type hypersensitivity into plasma in sufficient concentration to effect passive transfer of reactivity to tuberculoprotein or hen ovalbumin. But plasma from such animals did sensitize recipients for an Arthus reaction; and some of this early inflammation, both in actively and passively sensitized subjects, was still apparent at 24 hours. The significance of these late reactions is discussed in relation to the problem they pose when attempting to demonstrate a mediator of delayed-type hypersensitivity.     

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.

     

In vitro antigenic stimulation of peripheral blood and lymph node lymphocytes of sensitized guinea-pigs: the effect of a second administration of antigen in vivo

Stimulation of DNA synthesis by a guinea-pig albumin—orthanilic acid conjugate (AO) and by tuberculin purified protein derivative (PPD) was obtained in in vitro cultures of peripheral blood and lymph node lymphocytes from guinea-pigs with delayed hypersensitivity to these antigens.

Animals sensitized to both AO and PPD were given a further injection of 5 μg AO, intravenously, 8 hours before killing for in vitro studies. In these guinea-pigs, peripheral blood cultures, but not lymph node cultures, showed greater DNA synthesis in response to both AO and PPD than cultures from controls not given a further injection of AO.

It is suggested that the further increase in DNA synthesis was due to non-specific lymphocyte `activation' following the interaction of antigen and specifically sensitized lymphocytes.

     

The passive transfer of delayed hypersensitivity in the guinea-pig: I. The synergic effect of immune cells and immune serum on the 24-hour skin reaction and a study of the histology

Donor guinea-pigs were immunized with antigen in Freund's complete adjuvant. Three weeks later these donor guinea-pigs showed strong delayed hypersensitivity to the antigen. Peritoneal exudate cells (macrophages) and serum were then transferred intravenously from the immunized donors to normal recipients. Good 24-hour skin reactions were obtained in the recipients which had received cells and serum from donors immunized with bovine serum albumin, γ-globulin, ovalbumin and haemocyanin. The reaction transferred by immune cells and serum was greater than the reaction transferred by either singly. This synergy was not found with PPD or blood group substance as antigen. Good transfers of 24-hour skin reactions to PPD were obtained with peritoneal exudate cells both alone and with serum. Good transfers could not be obtained with blood group substance.

The synergic action of cells and serum in the passive transfer of 24-hour skin reactions was specific. The ability of peritoneal exudate cells to transfer 24-hour skin reactions was present at 1 week and greater at 3 weeks. In contrast the ability of serum to enhance passive transfers was absent at 1 week and present at 2 and 3 weeks.

The histology of reactions passively transferred by spleen, lymph node and peritoneal exudate cells in the presence and absence of serum was studied. Serum alone produced a predominantly polymorphonuclear infiltrate which was maximal at 18–24 hours and almost absent at 48 hours. Cells alone produced only a very slight reaction. The combination of cells and serum produced lesions at 4 and 18 hours resembling those caused by serum alone. At 24 hours however the histiocytes and lymphocytes were more numerous than in the pure serum reaction and by 48 hours the infiltrate consisted of histiocytes and lymphocytes.

It was concluded that, with certain antigens, there is a synergic effect of immune cells and serum in the passive transfer of 24-hour skin reactions and reasons are given for considering that these passively transferred 24-hour skin reactions are indeed delayed hypersensitivity reactions.

     

The Loss of Macrophages from Peritoneal Exudates following the Injection of Antigens into Guinea-Pigs with Delayed-Type Hypersensitivity

Exudates were induced in the peritoneal cavities of guinea-pigs by the injection of glycogen. The cell content of these exudates was examined after 4 days in normal and hypersensitive animals. In animals uninjected with antigens, the exudates contained a high proportion of macrophages, together with lymphocytes and polymorphs. In BCG-vaccinated animals, with delayed-type hypersensitivity to tuberculin, subcutaneous, intravenous or intraperitoneal injection of tuberculin resulted in a profound fall in the macrophage content of the exudates. This effect was apparent within an hour of intraperitoneal injection and occurred with very small doses of tuberculin. No such effect occurred after the intraperitoneal injection of tuberculin into guinea-pigs with Arthus hypersensitivity to tuberculin. Ovalbumin injected intraperitoneally into guinea-pigs with mixed delayed-type and Arthus hypersensitivity to ovalbumin also resulted in a marked fall in the macrophage content of peritoneal exudates, but had no effect on the peritoneal macrophages of animals with pure Arthus hypersensitivity. Bacterial endotoxin injected intraperitoneally caused a similar fall in the macrophage content of exudates of both normal and BCG-vaccinated animals. It is concluded that this loss of macrophages from peritoneal exudates after the injection of antigen is the consequence of an immunological reaction which is a manifestation of a state of delayed-type hypersensitivity.     

Production of delayed hypersensitivity by antigen associated with peritoneal exudate cells and the effect of pretreatment with Freund's complete adjuvant

The immunogenicity of antigens associated with peritoneal exudate cells was compared with the immunogenicity of free antigens for the induction of delayed hypersensitivity. Oil induced peritoneal exudate cells were incubated with antigens labelled with radioactive iodine, washed and injected intraperitoneally into recipient guinea-pigs. Comparable amounts of free antigens were also injected. All sixteen inbred guinea-pigs given human or bovine gamma-globulin associated with peritoneal exudate cells gave positive reactions (greater than 5 mm) while the eighteen guinea-pigs given free antigens failed to show a reaction. Similar results were obtained with serum albumins in inbred guinea-pigs. The increased immunogenicity of antigens associated with peritoneal exudate cells was also seen in outbred guinea-pigs.

Pretreatment with Freund's complete adjuvant did not depress the immunogenicity of antigens associated with peritoneal exudate cells and actually increased the induration of the 24-hour reaction to picrylated human serum albumin and human serum albumin associated with peritoneal exudate cells.

     

The influence of norepinephrine upon the histological pattern of the skin tuberculin reaction in the guinea-pig

The histological pattern of the tuberculin test in sensitized guinea-pigs was significantly influenced by simultaneously locally administered norepinephrine. Tissue lesions in the form of muscle necroses were registered even in deep layers of the subcutaneous muscle 3 hours later, at first without any inflammatory infiltration in the vicinity. This appeared only after considerable delay.

The time relation between the histologically demonstrable tissue lesions and the development of the inflammation indicates the primary importance of tissue damage in the pathogenesis of the cellular (delayed) hypersensitivity reaction.

     

Studies on the pathogenesis of experimental autoimmune renal tubulointerstitial disease in guinea-pigs. IV. failure to inhibit mononuclear cell accumulation with niridazole.

Niridazole is a potent inhibitor of certain expressions of delayed-type hypersensitivity; however, its effect and specificity on other immunologically induced mononuclear cell infiltrates has not been determined. We therefore tested this drug in a model of auto-antibody-induced renal tubulointerstitial disease (RTD), which is characterized by the accumulation of lymphocytes and macrophages in the cortical interstitium. Niridazole-treated (100 mg/kg) and untreated guineapigs were injected with potent anti-tubular basement membrane serum from donors with RTD. Drug treatment was repeated 2-3 times during the course of 10 days. The characteristic renal lesions were found in both groups. However, a single dose of niridazole inhibited skin reactivity in tuberculin sensitive guinea-pigs for at least 8 days. These findings further support the contention that immunologic tissue injury in RTD does not involve mechanisms of delayed-type hypersensitivity.     

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.

     

Radioautographic study of cellular mechanisms in delayed hypersensitivity: IV. Distribution of injected lymph node, spleen, thymus and bone marrow cells

Mononuclear cells from various organs of sensitized donor rats were labelled in vitro with tritiated leucine and injected intravenously into sensitized, syngeneic recipients. The localization of injected cells was studied in radioautographs.

Bone marrow cells were the most frequent of the labelled cells in skin reactions. Equal numbers of bone marrow cells were found in specific and non-specific delayed reactions and irritation reactions induced with turpentine.

Lymph node cells were found in delayed but not in turpentine reactions. Spleen cells were less frequent than lymph node cells in delayed skin reactions; small numbers were found in the turpentine reactions. Lymph node and spleen cells did not show detectable antigenic specificity.

Thymus cells were not found in the skin sites, even when they were allowed to circulate 2–3 days in the recipients before biopsies were taken.

Irrespective of the source of the cells injected few or no labelled cells were found in the recipient thymuses. Lymph node and spleen cells migrated equally into lymph nodes and spleen, but were infrequent in bone marrow. Only bone marrow cells had a decided preference for their organ of origin.

     

Eosinophilia: I. Eosinophilia in guinea-pigs mediated by passive anaphylaxis and by antigen—antibody complexes containing homologous IgG1a and IgG1b

There was an eight- to eleven-fold increase in the number of eosinophils in the peritoneal cavity of guinea-pigs repeatedly sensitized and subsequently challenged by that route. Peripheral blood eosinophilia was less and inconsistent. There were fewer eosinophils in the peritoneal cavity of guinea-pigs challenged intraperitoneally but sensitized by other routes, than in animals both sensitized and challenged peritoneally. Thus eosinophils were attracted to sites of antigenic challenge in anaphylactically sensitized tissue, particularly when sensitization occurred in the same tissue. In tissues repeatedly injected with antigen, there was an increase of mononuclear cells preceding and greater than the number of eosinophils.

The relation between eosinophilia and anaphylaxis was substantiated by evoking eosinophilia in normal guinea-pigs challenged after passive sensitization with homologous IgG1a (conferring PCA sensitivity for 3 to 4 days), and with IgG1b (conferring PCA sensitivity for 7 to 9 days) which mediate anaphylaxis, but not in animals treated with IgG2, which does not. Similarly complexes containing IgG1 antibodies evoke eosinophilia in normal animals, but complexes containing IgG2 do not.

Despite the association of eosinophilia with anaphylaxis, the number of peritoneal eosinophils following challenge is not greatly influenced by the susceptibility to fatal anaphylaxis, or by the serum PCA titre. Moreover it is doubtful whether passive sensitizing antibody mediates all the changes evoking eosinophilia on challenge, because guinea-pigs passively sensitized with IgG1 antibody for 16 hours had less eosinophilia on challenge than actively sensitized animals, though both passively and actively sensitized animals had the same titre of PCA antibody.

     

Response of human blood lymphocytes to tuberculin PPD in tissue culture

Human lymphocyte suspensions containing more than 99.2 per cent small lymphocytes were prepared from peripheral blood using methyl cellulose and carbonyl iron. The human donors were skin tested with tuberculin PPD or tuberculin OT. Their lymphocytes were cultured in the presence of tuberculin PPD and the transformed cells which appeared in the cultures were accurately quantitated.

When the corrected indices of response of lymphocyte cultures prepared from sensitized donors were plotted against the increasing amounts of tuberculin PPD used as a stimulus, a dose—response curve was obtained. The dose which gave the greatest distinction between lymphocytes from sensitized and from non-sensitized donors was 250 OT units of tuberculin PPD per ml of culture containing 2 × 10⁶ small lymphocytes per ml. Analysis of the culture population at 20-hour intervals for 100 hours showed that the transformed cells seen in vitro were derived from small lymphocytes.

Study of the behaviour of the stimulated cultures indicated that a 90-hour culture period was the most satisfactory for the accurate assessment of the immunological response.

A correlation was established between the immunological status of the lymphocyte donor and the corrected index of response of his lymphocytes in vitro. Cultures from non-sensitized donors had a corrected index of response close to zero. Corrected indices of response from sensitized donors ranged from 2 to 94 depending on the sensitivity of the donor to tuberculin PPD.

     

EXPERIMENTAL STUDY ON THE MECHANISMS OF DELAYED HYPERSENSITIVITY REACTION,WITH SPECIAL REFERENCE TO EMIGRATION and FUNCTION OF MONONUCLEAR CELLS*

Delayed hypersensitivity reaction is characterized by Infiltration of mononuclear cells (macrophages and lymphocytes). The mechanisms of emigration and function of mononuclear cells in local skin lesions were studied in experimental tuberculosis. A chemotactlc activity for mononuclear cells was found in the extract of tuberculin skin sites, and proteases active at pH 3, 6, and 9 were found in the extract and partially purified using ammonium sulfate and column chromatography. Relationship between chemotactlc activity and protease activities was discussed. Similar protease activities were found in the extract of sensitized lymph node cells, and proteases active at pH 6 and 9 were increased when homo-genate of sensitized lymph node cells was Incubated with PPD for 3 hours at 37°C. It appears that PPD could concern to release protease from sensitized lymphocytes and involve in delayed hypersensitivity reactions. The role of mononuclear cells in tuberculous skin lesions were studied in the sense of bacteriocidal activity for tubercle bacilli. Certain macrophages in the skin lesions were produced locally by cell division. Macrophages in granulation tissue around necrotic foci were specifically matured and strongly active for/9-galactosidase, and the number of acid fast bacilli in macrophages and the intensity of staining for /3-galactosidase were reversely correlated. This was also confirmed in irradiated rabbits with 400 rads of whole body radiation. These findings suggest that high levels of lysosomal enzymes are involved in destruction of bacilli.     

Interaction between `sensitized lymphocytes' and antigen in vitro: I. The release of a skin reactive factor

Peritoneal exudate lymphocytes (PEL), purified on glass beads and lymph node (LN) cells from guinea-pigs immunized with tubercle bacilli were cultured for 24 hours, in serum-free medium, without and with various concentrations of Tuberculin PPD.

Supernatants obtained from cultures with 10 μg PPD/10⁷ lymphocytes provoked an intense inflammatory reaction, when injected into the skin of normal guinea-pigs. PEL were more active than LN cells from the same animals. The reaction was characterized by erythema and induration, with a peak between 3 and 6 hours and histologically a mixed polymorphonuclear—mononuclear infiltrate in the dermis was seen. When fractionated on Sephadex G-200, skin activity of both PEL and LN supernatants was concentrated in a peak corresponding to the molecular weight of serum albumin, while in LN material some activity was also present in a small molecular weight peak. The active material could be separated from albumin by chromatography on DEAE-cellulose. Immunoelectrophoretic analysis of skin reactive peaks detected a slow α-globulin in both PEL and LN supernatants. PPD in the form of a complex with a protein precipitated by anti-γG antiserum, was detected in the skin-active Sephadex peak III of LN supernatants, by radioimmunoelectrophoresis. Skin activity was precipitated with ammonium sulphate at 66 per cent saturation and was destroyed by pepsin treatment. Formation of the skin-active material was depressed by Puromycin and Actinomycin-D and the development of skin inflammation was suppressed by pretreatment of the recipient with anti-lymph node extract serum.

Evidence for antigen induced specific synthesis and release of an α-globulin in PEL and LN cultures was found but its relation to the skin active material is unknown.

     

Fluorescent Protein Tracer Studies in Allergic Reactions: I. The Fate of Fluorescent Antigen in Active and Passive Arthus Reaction in the Guinea-Pig Skin

The fate of intradermally injected bovine serum albumin, labelled with lissamine rhodamine was studied in actively and passively sensitized guinea pigs, and compared with the fate of the same protein in control animals. In the latter, the antigen is taken up by macrophages, apart from a discharge of antigen to the regional lymph nodes.

In the sensitized animals, in which severe Arthus reactions occurred, an accumulation of antigen in vessel walls and lumina of capillaries and venules was observed. Polynuclear leucocytes take antigen after a few hours. This is a specific reaction absent with simultaneously injected protein to which the animal is not sensitized.

     

Experimental immune inflammation in the synovial membrane: I. The immunological mechanism

Synovitis was produced in immunized guinea-pigs and rats by injection of antigen into the knee joint. The reaction was mainly mononuclear at 48 hours and, in the guinea-pig, progressed to chronic granuloma formation. Antigens principally used were bovine γ-globulin and tuberculin PPD. Immune deviation, giving a diminished delayed hypersensitivity response, also gave diminished synovial inflammation when compared with undeviated control animals. Immune synovitis to tuberculin PPD was successfully transferred with peritoneal cells taken from immunized animals and given intravenously to normal recipients whose knee joints were injected with antigen. Intravenous transfer of immune serum gave rise to a synovial reaction to bovine γ-globulin, injected into recipients' joints. Transfer of both cells and serum gave rise to particularly severe reactions. Transfer of either cells or serum or cells with serum failed to give reactions extending beyond 3–4 days. This suggests that active immunization is a requisite for the chronic inflammatory reaction of synovitis. Although the 48-hour synovial membrane reaction of rats was similar to that seen in the guinea-pig, chronic inflammatory reactions were not found in that species.     

The Effect of an Unrelated Delayed-Type Hypersensitivity Reaction on the Antibody Response to Diphtheria Toxoid

Four Lf fluid diphtheria toxoid were injected intracutaneously, together with tuberculin PPD, into guinea-pigs sensitized to tuberculin by a water-in-oil emulsion of killed tubercle bacilli 4–5 months previously. Antibody production, judged by antibody levels at 4 weeks, was two to four times greater than that in control guinea-pigs. Such an increase in antitoxin would correspond to a ten- to twelve-fold increase in the effective dose of toxoid, which is much greater than any likely non-specific retention at the injection site. It is suggested that the effect of a local tuberculin reaction in enhancing the immunogenic capacity of diphtheria toxoid is due to an increase in the number of immunologically competent cells to which the toxoid has access.