The effect of irradiation on the passive transfer of delayed hypersensitivity

Delayed hypersensitivity was passively transferred in guinea-pigs by lymph node and peritoneal exudate cells. This transfer was unaffected by irradiation with 1000 r given in vitro but was reduced by 1500, 2000 and 3000 r.     

The induction of delayed hypersensitivity by macrophage-associated antigen: The role of macrophage cytophilic antibody

The regulation of the immune responses by antibody administered passively on peritoneal exudate cells (PEC) was studied in guinea-pigs. The immunogenicity of sheep red blood cells (SRBC) associated with PEC for the induction of delayed hypersensitivity (DH) to soluble erythrocyte antigen was enhanced when PEC were incubated with anti-SRBC antibody. In contrast, the antibody response to SRBC was depressed. This phenomenon was only observed with specific antibody and was partially blocked when PEC covered with anti-SRBC antibody were incubated with rabbit anti-guinea-pig globulin serum. Comparable amounts of anti-SRBC antibody injected separately had no enhancing effect. Anti-SRBC sera from which the cytophilic antibody activity had been removed were inactive. PEC-associated SRBC were usually more immunogenic than the same amount of `free' SRBC for the induction of delayed hypersensitivity and always more immunogenic for antibody production.

These observations suggested that macrophage cytophilic antibody might be involved in the regulation of the immune response and play a role in the preferential induction of delayed hypersensitivity in the studied system.

     

Local passive transfer of delayed type hypersensitivity to syngeneic testicular cells in mice.

A means for local passive transfer (LPT) of delayed footpad reaction (DFR) against syngeneic testicular cells (TC) was established in mice. Peritoneal exudate (PE) cells from immunized donors pre-treated with cyclophosphamide (Cy) were transferred with syngeneic TC into a hind footpad of a naive mouse. Antigen specific DFR was successfully transferred by PE cells as early as 3-5 days after immunization and as late as 39 days after immunization. DFR was abolished after treatment of PE cells with anti-0 serum, indicating T cell dependence. No footpad reaction can be elicited in male mice immunized with syngeneic TC without Cy pre-treatment. However, PE cells of those mice can passively transfer footpad reaction locally, suggesting the existence of suppressive mechanism in the donors. Although no suppressive mechanism was disclosed at the present, this LPT system seems a potent tool for further analysis of the regulatory mechanisms.     

The mechanism of anaphylaxis: II. The role of `mast cell sensitizing' antibody and delayed hypersensitivity in rat anaphylaxis

Both immediate and delayed cutaneous reactions were found to be present in rats actively sensitized by the injection of antigen plus Bordetella pertussis organisms. Delayed hypersensitivity preceded the appearance of `mast cell sensitizing' antibody (MCSAb) in those animals which were only fully susceptible to anaphylaxis when both delayed and immediate reactions had reached their maximum. Passive anaphylaxis with MCSAb was fatal only when transferred to actively sensitized rats at a time when the animals had delayed cutaneous reactions but were not yet fully susceptible to anaphylaxis. It is suggested that anaphylaxis in rats is due to an additive or synergistic effect between immediate and delayed hypersensitivities.     

Contact and delayed hypersensitivity in the mouse: I. Active sensitization and passive transfer

Ear swelling in mice was measured with a micrometer and used to quantify 24-hour skin reactions. Specific contact sensitivity occurred in mice immunized with picryl chloride, 2-phenyl-4-ethoxymethylene oxazolone, dinitrofluorobenzene and tetramethyl-p-phenylenediamine as shown by ear swelling 24 hours after challenge. In some mice sensitized with oxazolone significant swelling occurred 4 hours after challenge. It was possible to induce tolerance in adult mice and contact sensitivity to dinitrochlorobenzene was diminished by prior treatment with dinitrobenzenesulphonic acid.

There was some evidence of delayed hypersensitivity to protein antigens. Twenty-four-hour skin reactions to PPD (purified protein derivative of tuberculin) occurred in mice immunized with live BCG or dead tubercle bacilli in Freund's adjuvant. In most experiments the swelling at 24 hours was greater than at 4 hours. Similar reactions to egg albumin and bovine γ-globulin were seen in mice immunized with these antigens in Freund's complete adjuvant and one group of mice showed greater swelling at 24 hours after challenge than at 4 hours.

Contact sensitivity was readily transferred in inbred mice by peritoneal exudate cells when the challenge was undertaken immediately after transfer. Variable and usually inferior passive transfer was obtained with lymph node cells. Attempts to transfer contact sensitivity in outbred mice were unsuccessful.

     

Contact and delayed hypersensitivity in the mouse: II. The role of different cell populations

Mice sensitized to oxazolone show contact sensitivity which can be assessed by painting oxazolone on the ear and measuring the increase in ear thickness at 24 hours. Contact sensitivity can be transferred passively by peritoneal exudate, lymph node and bone marrow cells. When CBA mice are challenged immediately after transfer, at a 3:1 donor recipient ratio, the peritoneal exudate cells give bigger transferred reactions than lymph node cells. The converse is true on challenge at 6 days after transfer, at which time the lymph node cells give bigger transferred reactions than the peritoneal exudate cells. These results suggest that different types or functional states of cells are involved in these two types of transfer.

The possibility that the successful transfer with delayed challenge by lymph node cells was due to active sensitization by antigen transferred with the cells, was made unlikely by passive transfer into `tolerant' recipients.

Mice first show contact sensitivity when challenged at 3 days (about 72 hours) after sensitization. The lymph nodes develop the ability to transfer contact sensitivity with delayed challenge at the same time but the bone marrow only acquires this activity at 5 days.

     

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.     

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.

     

A radioisotopic method to measure delayed type hypersensitivity in the mouse. II. Cell transfer studies.

Sensitized lymphoid cells could transfer to normal non-sensitized (naive) mice by 24-48 h after antigen challenge in the ear, the capacity to incorporate, at the site of antigen deposition, 5-iodo-2'deoxyuridine-125I in amounts significantly above those obtained in control mice. This was associated with a mononuclear cell infiltration in the pinna. In contrast to lymphoid cells, serum antibodies were unable to transfer a 24- or 48 hour ear reaction. The cells responsible were T lymphocytes as demonstrated by successful transfer following enrichment for T lymphocytes, and abrogation of transfer following treatment with anti-theta serum and complement. Transfer was achieved whether the naive recipients were normal, T-cell deprived, pretreated with cyclophosphamide, or lightly irradiated but not when they were heavily irradiated. Adoptive transfer of the 24-hour ear response was demonstrated with three different antigenic systems. The time-response curves were different with each system although peak reactions were obtained 5 days after sensitization of the donors in all cases. The specificity patterns of the 24-hour ear reaction on transfer were similar to those obtained in the sensitized donors. The results of these studies indicate that the radioisotopic ear method can, under defined conditions, demonstrate the existence of a state of delayed type hypersensitivity in the donors.     

Differential ability of occupational chemical contact and respiratory allergens to cause immediate and delayed dermal hypersensitivity reactions in mice.

Trimellitic anhydride (TMA) is known to cause occupational respiratory allergy associated with the presence of specific IgE antibody. Other chemicals, such as 2,4-dinitrochlorobenzene (DNCB), while exhibiting a clear potential for contact sensitization, apparently lack the ability to induce respiratory allergy in man. It has been shown previously that although both chemicals are immunogenic in mice, each provoking contact sensitization, exposure only to TMA results in an IgE antibody response. In the present study, to examine further the characteristics of human allergens, we have compared the ability of TMA and DNCB to elicit immediate and delayed cutaneous hypersensitivity reactions in mice. Topical exposure to both chemicals resulted in delayed (24 h) hypersensitivity. However, only TMA induced, in addition, an immediate (1 h) dermal reaction following local challenge. Serum from TMA-immune mice, but not from untreated mice or mice sensitized with DNCB, was able to transfer immediate hypersensitivity to naive recipients. The kinetics of passive sensitization with TMA-immune serum, together with the fact that immediate hypersensitivity to DNCB could be induced with monoclonal IgE anti-dinitrophenol (DNP) antibody, suggests that the immediate dermal responses caused by TMA are effected by hapten-specific IgE. These data demonstrate that different classes of occupational chemical allergen exhibit a variable potential to elicit immediate and delayed dermal hypersensitivity reactions in mice, and provide a novel approach to the classification and characterization of human allergens.     

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.

     

Lymphocyte chemotaxis in inflammation. VIII. Demonstration of lymphocyte chemotactic lymphokines in PPD-induced delayed hypersensitivity skin reaction site in the guinea-pig.

The experiments were carried out to clarify whether lymphocyte chemotactic factors (LCFs) derived from activated lymphocytes, i.e. lymphocyte chemotactic lymphokines would exist in delayed-type hypersensitivity (DTH) reaction sites in guinea-pigs. To analyse the problem, we attempted to use an immunoadsorbent column conjugated with respective antibodies against LCFs (LCF-b, LCF-c and LCF-d) isolated from purified protein derivative (PPD)-induced DTH skin reaction sites in guinea-pigs. The chemotactic activity of culture supernatants from PPD- or concanavalin A (Con A)-stimulated lymph node (LN) cells was decreased to about 50% by the immunoadsorbent column with anti-LCF-c antibody or anti-LCF-d antibody, while its activity was little or not influenced by the columns with anti-LCF-b, anti-IgG or anti-IgM antibody. Further experiments using successive immunoadsorption with anti-LCF-c antibody followed by anti-LCF-d antibody showed almost the complete adsorption of the chemotactic activity in the above culture fluids. Additionally, the chemotactic lymphokine which was absorbed by anti-LCF-c antibody had a similar mol. wt. to that of LCF-c (mol. wt about 160,000). However, the chemotactic lymphokine which was absorbed by anti-LCF-d antibody had a mol. wt. of about 27,000; it was clearly distinct in mol. wt. from LCF-d (mol. wt. about 300,000). It is thus suggested that at least one of lymphocyte chemotactic lymphokines exists in the DTH reaction sites and functions as LCF-c.