(Received for publication, July 26, 1976)

Transcription

1 Microbiol. Immunol. Vol. 21 (2), , 1977 Relation between Delayed Skin Reactivity and Macrophage Migration Inhibition or Lymphocyte Transformation in Tuberculin-Type Hypersensitivity and Jones-Mote Hypersensitivity Kunisuke HIMENO, Kikuo NOMOTO, Ataru KUROIWA, SUMi0 MIYAZAKI, and Kenji TAKEYA Department of Microbiology, and Department of Pediatrics, Faculty of Medicine, Kyushu University, Fukuoka (Received for publication, July 26, 1976) Abstract Precise time-course studies on delayed skin reaction, lymphocyte transformation and macrophage migration inhibition were carried out from day 3 to 270 and from day 3 to 120, respectively, in guinea pigs immunized with bovine gamma-globulin (BGG) in complete Freund's adjuvant (CFA) and those immunized with BGG in incomplete Freund's adjuvant (IFA). a) Delayed skin reactions could be elicited for a long period of time after immunization with BGG in CFA in the presence of prominent antibody production and were accompanied by induration. b) Delayed reactions could be elicited transiently after immunization with BGG in IFA and were not accompanied by induration. c) At the peak of hypersensitivity, infiltrating cells at the reaction sites were composed largely of mononuclear cells and basophils, respectively, in the animals immunized with BGG in CFA and those immunized with BGG in IFA. d) Uptake of 3H-thymidine by lymphocytes was increased remarkably in the presence of BGG when cells were obtained at early stages after immunization by both methods. e) Macrophage migration inhibition was strongly positive in animals immunized with BGG in CFA but weakly positive in those immunized with BGG in IFA. Increased lymphocyte transformation preceded the appearance of a positive migration inhibition. f) After immunization with BGG in CFA, Jones-Mote hypersensitivity appeared to precede the development of tuberculin-type hypersensitivity. Delayed hypersensitivity includes several distinctive phenomena of which the common feature is a delayed onset of skin reactions after administration of the eliciting antigens. Classical delayed hypersensitivity of tuberculin-type, Jones-Mote hypersensitivity and contact sensitivity have been assigned to this category. However, mutual relationships among individual reactions and essential characteristics of each reaction have not yet been identified definitely. Lymphocyte blastoid trans- The results in this paper were reported at the Annual Congress of the Japanese Society for Bacteriology in 1975 and in the Proc. Japan. Soc. Immunol. Vol. 5 (1),

2 100 K. HIMENO ET AL formation (6, 14) and macrophage migration inhibition (4, 7) have been proposed as in vitro correlates of such delayed hypersensitivity. In many of the hosts showing positive skin reactions, lymphocyte transformation and macrophage migration inhibition can be detected concurrently (2, 8, 12). However, under some experimental conditions or in some clinical cases, these indicators of delayed hypersensitivity can be elicited in dissociated forms. Therefore, mutual relationships among these in vivo and in vitro reactions have to be understood more precisely for further advances in the studies on delayed hypersensitivity. In the present study, precise kinetics of in vivo skin reactions, in vitro reactions and antibody production in guinea pigs were observed after immunization with bovine gamma-globulin (BGG) in Freund's adjuvants. MATERIALS AND METHODS Animals. Female guinea pigs of an outbred Hartley strain were obtained from a local breeder. Adult animals of 350 to 450 g were used for experiments. Immunization. BGG (Cohn fraction II, Sigma) was used as an immunogen and a test antigen. A 5 mg/ml solution of BGG in saline was added to an equal volume of an oil phase containing 8.5 ml of Drakeol Six and 1.5 ml of Arlacel A (incomplete Freund's adjuvant, IFA). To prepare complete Freund's adjuvant (CFA), 50 mg of heat-killed, dried BCG was added to the oil phase. Samples of 0.2 ml of such emulsions were injected into each of four footpads. Consequently, each animal received 2.0 mg of BGG in CFA or IFA. Immune responses were assessed on three to four animals taken randomly from each group at various times after immunization. Two days before the injection of eliciting doses of BGG, liquid paraffin was injected intraperitoneally. Skin reactions were read 2 and 24 hr after the elicitation and the animals were sacrificed immediately after the measurement of the 24-hr reaction to obtain peritoneal exudate cells (PEC), lymph node cells and sera. Skin testing. Eliciting doses of 0.2 mg and 0.02 mg of BGG in 0.1 ml of saline were injected intradermally into the right and left sides of shaved dorsal flanks, respectively. Immediate reactions were read 2 hr after the injection of 0.2 mg of BGG, and delayed reactions were read 24 hr after the injection of 0.02 mg of BGG. Immediate reactions were graded as negative ( j, weakly positive (±) and positive (+) according to the degrees of erythema accompanied by hemorrhage. With respect to delayed reactions, the averages of the longest and shortest diameters of erythema and the presence or absence of induration were recorded. At the site injected with 0.02 mg of BGG, immediate reaction of the Arthus type was very weak even in the presence of strong immediate reaction at the site injected with 0.2 mg of BGG in the same animal. Macrophage migration inhibition test. The macrophage migration inhibition test was carried out according to the direct method of George and Vaughan (7) after minor modifications. Thirty milliliters of liquid paraffin were injected intraperitoneally and PEC were harvested 72 hr later. PEC were obtained by washing the peritoneal cavity with Hanks' balanced salt solution (HBSS) after complete bleeding.

3 BLASTOGENESIS AND DELAYED REACTION 101 Capillary tubes packed with PEC were secured with silicone grease on the surface of a cover glass which constituted the bottom of a culture chamber. Two holes of 15 mm in diameter in an acrylic plate (75 x 25 x 5 mm) were used as culture chambers. The chambers were filled with TC-199 (Difco) supplemented with 20% fresh guinea pig serum. The top of the hole was covered with a cover glass. BGG was added to the medium at a concentration of 1.0 mg/ml. The chambers were cultured in an atmosphere of 5% CO2 and 95% air for 24 hr at 37 C. Areas of migration were measured under an ocular micrometer on an inversion microscope. Percent migration inhibition (% MI) was calculated by the following formula: migration area in the presence of antigen migration area in the absence of antigen Assay of lymphocyte transformation. The axillary and inguinal lymph nodes were obtained immediately after sacrificing. The lymph nodes were cut into small pieces and squeezed with two slide glasses in HBSS to prepare cell suspensions. The cell suspensions were passed through two layers of gauze to remove large debris. The single-cell suspensions were adjusted to a concentration of 5 x 106 viable cells/m1 in TC-199 supplemented with 20% normal fresh guinea pig serum after counting viable cells by the trypan blue exclusion method. Two milliliters of the cell suspensions were dispensed into tissue culture tubes (Falcon 3033) and incubated in an atmosphere of 5% CO2 and 95% air at 37 C in the presence or absence of 1.0 mg/m1 of BGG. After incubation for 60 hr, 3H-thymidine was added to the cultures at a concentration of 2.0,uCi/ml. After additional incubation for 12 hr, the cell suspensions were harvested and incorporation of 3H-thymidine into lymphocytes was assessed by liquid scintillation counter (Beckman) and expressed as count per minute (cpm). The stimulation index (SI) was calculated by the following formula : cpm in the presence of antigen cpm in the absence of antigen Titration of antibody. Sera obtained at the time of sacrificing were examined for the presence of anti-bgg antibody using a passive hemagglutination test. BGG was coupled to formalin-fixed sheep erythrocytes by the bisdiazobenzidine method (1). Titration was carried out by the microtitration method. Histological examination. Reaction sites were removed from animals immediately after sacrificing. The pieces of full-thickness skin were fixed in 10% formalin solution. Paraffin sections were stained with hematoxylin-eosin and alkaline and acid Giemsa (5). RESULTS Skin Reactions after Immunization with BGG in CFA and IFA Sixty-five animals were immunized with BGG in CFA and three or four animals each were taken for the elicitation of skin reactions 2, 4, 6, 8, 10, 12, 14, 16, 20, 27, 44, 59, 89, 119, 149, 179, and 269 days after immunization (Fig. 1, left). Immediate reactions (2 hr) were measured on the right sides injected with 0.2 mg of BGG and delayed reactions (24 hr) were measured on the left sides injected with 0.02 mg of

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6 104 K. HIMENO ET AL BGG. Delayed skin reactions became detectable on day 4, but those on days 4 and 6 were not accompanied by induration. Induration became detectable on day 8. Positive deayed reactions accompanied by induration were detected by the last day of observation, i.e., day 269. Weakly positive reactions of the Arthus type were elicited from days 8 to 12. Strongly positive reactions of the Arthus type were detectable from days 14 to 269. Fifty animals were immunized with BGG in IFA and three or four animals each were taken for the elicitation of skin reactions 2, 4, 6, 8, 10, 12, 14, 16, 20, 27, 44, 89, and 119 days after immunization (Fig. 1, right). Delayed skin reactions were detected from days 4 to 16 and they became undetectable on day 20 and thereafter. Positive reactions of the delayed type showed erythema but were not accompanied by induration at any time. Immediate reactions showed a pattern similar to those in the animals immunized with BGG in CFA. In Vitro Delayed Reactions and Antibody Titers in Animals Immunized with BGG in CFA Cellular specimens and sera were obtained from the animals shown in Fig. 1 immediately after measurement of 24-hr skin reactions. SI was slightly increased in the lymph node cells obtained on day 3 or 5 of immunization with BGG in CFA as compared to SI in non-immunized controls (Fig. 2). A tremendously augmented SI was detected in the lymph node cells obtained on day 7 of immunization. Lymph node cells obtained thereafter showed weak increases in SI. Macrophage migration Fig. 3. Time-course studies on lymphocyte transformation (., macrophage migration inhibition (0) and hemagglutinating antibody (A) in guinea pigs immunized with BGG in IFA. Non-immunized guinea pigs were used as controls. Specimens were obtained immediately after the measurement of 24-hr skin reactions. Dates in figure indicate the days of harvesting cellular and humoral specimens. Bars represent ranges.

7 BLASTOGENESIS AND DELAYED REACTION 105 was strikingly enhanced by the addition of BGG when PEC were obtained on day 3 % MI : ) or day 5 (% MI : ) of immunization. Migration was slightly inhibited when PEC were obtained from day 7 to 11. Migration was inhibited to remarkable extents in the presence of BGG when PEC obtained on day 15 or later were used. Anti-BGG antibody became detectable in the sera obtained on day 9 (Fig. 2). The sera obtained on day 15 or later showed a plateau at a very high level of antibody titer. A major portion of the antibodies detected on day 9 or later was composed of antibodies resistant to treatment with 2-mercaptoethanol. In Vitro Delayed Reactions and Antibody Titers in Animals Immunized with BGG in IFA Cellular specimens and sera were obtained from the animals shown in Fig. 1 immediately after measurement of 24-hr skin reactions. SI increased substantially when lymph node cells were obtained on day 5 or 7 of immunization with BGG in IFA (Fig. 3). Lymph node cells obtained on day 9 or later exhibited no or very low increases in SI as compared to that in non-immunized controls. Macrophage migration was inhibited to moderate extents by the addition of BGG when PEC were obtained from days 9 to 17. When PEC obtained on day 21 or later were used, migration was not inhibited in the presence of BGG. Migration was enhanced slightly by the addition of BGG when PEC were obtained on day 3 (% MI : ) and on day 90 (% MI : - 0.0) of immunization. Anti-BGG antibody became detectable in the sera obtained on day 5 and its titers on day 11 increased to reach a plateau of aconsiderably high level. The major portion of antibodies detected on day 9 or later was composed of antibodies resistant to treatment with 2-mercaptoethanol. Histological Examinations of Skin Reaction Sites Skin reaction sites were removed immediately after the measurement of delayed skin reactions from the animals elicited with BGG 6 or 20 days after immunization (Fig. 4). When skin reactions were elicited 20 days after immunization with BGG in CFA, a well-known, typical feature of tuberculin-type hypersensitivity was revealed. An extensive cellular infiltration was detected in the dermis and the infiltrating cells were composed of mononuclear cells (35 to 50%), basophils (10 to 20%), eosinophils (10 to 20%) and neutrophils (10% or less). When skin reactions were elicited 6 days after immunization with BGG in IFA, a typical feature of Jones-Mote hypersensitivity was revealed. An extensive cellular infiltratin was detected in the dermis, just as that at the reaction sites of tuberculin-type hypersensitivity. However, the infiltrating cells were composed largely of basophils (60 to 80%). Substantial numbers of mononuclear cells (15 to 30%) and small numbers of eosinophils and neutrophils were detectable at the reaction sites. When skin reactions were elicited 6 days after immunization with BGG in CFA, the majority of infiltrating cells were composed of basophils, just as at the reaction sites of the animals immunized with BGG in IFA.

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9 BLASTOGENESIS AND DELAYED REACTION 107 Fig. 4. Histological features of the upper dermis in delayed skin hypersensitivities. (A) Tuberculin hypersensitivity elicited with BGG 20 days after immunization with BGG in CFA. Mononuclear cells comprised the majority of infiltrating cells. (B) Jones-Mote hypersensitivity elicited 6 days after immunization with BGG in IFA. Basophils comprised the majority of infiltrating cells. (C) The accumulation of basophils at the reaction sites elicited 6 days after immunization with BGG in CFA. Sectionswere stained with alkaline Giemsa solution. x 830 DISCUSSION Delayed erythematous skin reactions can be elicited in guinea pigs immunized with protein antigens in IFA or antigen-antibody complexes and they have been called Jones-Mote reactions to differentiate them from classical tuberculin-type hypersensitivity (15, 17, 18). Jones-Mote hypersensitivity can be elicited only at an early period of such immunization and becomes undetectable with the prominent appearance of serum antibodies. Thus, identification of this type of delayed reaction as a distinct entity has been difficult for a long time since the first report on human subjects by Jones and Mote (9), and many investigators have regarded Jones-Mote reactivity as a weak expression of delayed hypersensitivity. Recently, Dvorak and his coworkers demonstrated that extensive infiltration of basophils at the reaction sites was the most discriminative feature ofjones-mote reactions (5). The results given in this paper comfirmed the discrimination between tuberculin hypersensitivity and Jones-Mote hypersensitivity presented by other investigators (3, 10, 17, 18) in that

10 108 K. HIMENO ET AL a) tuberculin-type hypersensitivity persisted for a long period after immunization with BGG in CFA in the presence of prominent antibody production, and skin reactions were accompanied by induration; b) the Jones-Mote reaction could be elicited only transiently after immunization with BGG in IFA, and skin reactions were not accompanied by induration; c) at their fully developed stages, infiltrating cells at the reaction sites were composed largely of mononuclear cells in animals immunized with BGG in CFA and of basophils in animals immunized with BGG in IFA. Macrophage migration inhibition has been considered as an in vitro correlate of cellular immunity including various types of immunological reactions. Recently, macrophage migration inhibition was reported to be an in vitro correlate of tuberculintype hypersensitivity but not of Jones-Mote hypersensitivity. In the experiment by Katz et al (10), the macrophage migration inhibition was strongly positive when PEC were obtained from guinea pigs immunized with ovalbumin in CFA. However, migration inhibition was negative when PEC were obtainedfrom guinea pigs immunized with the antigen in IFA. In the experiment in our laboratory (13), the macrophage migration inhibition was positive when PEC were obtained from mice immunized with sheep erythrocytes (SRBC) in CFA or those pretreated with BCG and then immunized with SRBC in saline. When mice were immunized with SRBC in saline alone, macrophage migration was not inhibited by the addition of SRBC antigen. Even when delayed footpad reactions were remarkably enhanced by pretreatment of the mice with cyclophosphamide, migration inhibition was negative. Also in the present study, macrophage migration was inhibited strikingly by the addition of BGG with respect to the PEC obtained from guinea pigs immunized with the antigen in CFA. However, macrophage migration was also inhibited weakly but definitely at the peak of skin reactions after immunization with BGG in IFA. Although the meaning of a weakly positive inhibition of macrophage migration in such animals can not be understood definitely at the present time, the presence or absence of a positive migration inhibition appears to be helpful but not conclusive in discrimination of both types of delayed hypersensitivity at least in the guinea pig. Jones-Mote hypersensitivity appears to precede the appearance of tuberulin hypersensitivity at early stages after immunization with BGG in CFA. When delayed skin reactions were elicited on day 4 or 6 in guinea pigs immunized with BGG in CFA, erythematous reactions were not accompanied by induration. Basophils comprised the majority of infiltrating cells at such reaction sites as reported by Dvorak and his co-workers (5). When PEC obtained from such animals were used, macrophage migration was not inhibited by the addition of the antigen. These findings may be compatible with those for Jones-Mote hypersensitivity but not for tuberulintype hypersensitivity. Uptake of 3H-thymidine by lymphocytes was increased to a tremendously high degree in the presence of BGG in the culture medium when lymph node cells were obtained at an early stage after immunization with BGG in CFA. At such a stage, migration inhibition was only weakly positive. At later stages when migration inhibition became strongly positive, lymphocyte transformation in the presence of BGG was slightly enhanced as compared to that in non-immunized controls. Thus,

11 BLASTOGENESIS AND DELAYED REACTION 109 macrophage migration inhibition and lymphocyte transformation may represent different phases of delayed hypersensitivity. Strinkingly increased uptake of 3Hthymidine in the presence of BGG was detectable at an early stage after immunization with BGG in IFA. In these animals, the rate of lymphocyte transformation became very low also at later stages. Therefore, a striking increase in 3H-thymidine uptake by lymph node cells in the early stages may represent a proliferative phase of the sensitized lymphocytes which precedes the full development of delayed hypersensitivity. At later stages, proliferation of lymphocytes may be required only for the supply of sensitized lymphocytes to maintain the levels of positive delayed hypersensitivity. This explanation may be compatible with the report by Ricci et al (16). In their experiment on guinea pigs immunized with CFA, in vitro transformation of the peripheral blood lymphocytes in response to PPD was related closely to the appearance of delayed skin reactions, while migration of peritoneal cells was markedly inhibited only at later stages of immunization when much evidence of strong in vivo and in vitro reactions of delayed hypersensitivity became detectable. Dissociation between lymphocyte transformation and production of chemical mediators has been reported under several experimental and clinical conditions accompanied by some immunodeficiencies (11, 19). Such dissociations have to be analyzed from the viewpoint of kinetics of immune responses as presented in this paper. Our results indicate that several kinds of in vivo and in vitro tests have to be carried out on the same individuals to determine their exact immunological states. In the present study, specimens for in vitro assays were obtained 24 hr after the elicitation of skin reactions and it might be doubted that the eliciting dose of the antigen exerts effects on the resposiveness to be estimated by in vitro assays. However, in vitro assays carried out on guinea pigs immunized but not given an eliciting dose proved that the elicitation exerted no detectable effects on such in vitro responsiveness. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1) Campbell, D.H., Garvey, J.S., Cremer, N.E., and Sussdorf, D.H Methods in immunology, p , 2nd ed, W.A.Benjamin Inc., New York. 2) Chaparas, S.D., Sheagren, J.N., Demeo, A., and Hedrick, S Correlation of human skin reactivity with lymphocyte transformation induced by mycobacterial antigens and histoplasmin. Amer. Rev. Resp. Dis. 101: ) Coe, J.E., and Salvin, S.B The immune response in the presence of delayed hypersensitivity or circulating antibody. J. Immunol. 93: ) David, J.R., Al-Askari, S., Lawrence, H.S., and Thomas, L Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93: ) Dvorak, H.F., Dvorak, A.M., Simpson, B.A., Richerson, H.B., Leskowitz, S., and Karnovsky, M. J Cutaneous basophil hypersensitivity. II. A light and electron microscopic description. J. Exp. Med. 132: ) Elves, M.W., Roath, S., and Israels, M.C.G The response of lymphocytes to antigen challenge in vitro. Lancet i: ) George, M., and Vaughan, J.H In vitro cell migration as a model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111:

12 110 K. HIMENO ET AL 8) Hinz, C.F., Daniel, T.M., and Baum, G.L Quantitative aspects of lymphocytes by tuberculin purified protein derivative. Int. Arch. Allergy 38: ) Jones, T.D., and Mote, J.R Phases of foreign protein sensitization in human beings. New England J. Med. 210: ) Katz, S.I., Parker, D., and Turk, J.L Mechanisms involved in the expression of Jones- Mote hypersensitivity. II. Lymph node morphology and in vitro correlates. Cell. Immunol.16: ) Kirchner, H., Altman, L.C., Fridberg, A.P., and Oppenheim, J. J Dissociation of in vitro lymphocyte transformation from production of a monocuclear leucocyte chemotactic factor in agammaglobulinemic children. Cell. Immunol. 10: ) Matsaniotis, N.C., Tsenghi, C., Economou-Mavrou, C., and Metaxotou-Stavridaki, C Skin reactivity and in vitro lymphocyte reactivity to tuberculin in childhood. J. Pediat. 72: ) Ohmichi, Y., Nomoto, K., Yamada, H., and Takeya, K Relationships among differentiated T-cell subpopulations. I. Dissociated development of tuberculin type hypersensitivity, Jones-Mote hypersensitivity and activation of helper function. Immunology 31: ) Pearmain, G.E., Lycette, R.R., and Fitzgerald, P.H Tuberculin-induced mitosis in peripheral blood leucocytes. Lancet i: ) Raffel, S., and Newel, J.M The "delayed hypersensitivity" induced by antigen-antibody complexes. J. Exp. Med. 108: ) Ricci, M., Romagnani, S., Passaleva, A., and Biliotti, G Lymphocyte transformation and macrophage migration in guinea pigs immunized with Freund's complete adjuvant. Clin. Exp. Immunol. 5: ) Salvin, S.B Occurrence of delayed hypersensitivity during the development of Arthus type hypersensitivity. J. Exp. Med. 107: ) Uhr, J.W., Salvin, S.B., and Pappenheimer, A.M., Jr Delayed hypersensitivity. II. Induction of delayed hypersensitivity in guinea pigs by means of antigen-antibody complexes. J. Exp. Med. 105: ) Valdimarsson, H., Higgs, J.M., Wells, R.S., Yamamura, M., Hobbs, J.R., and Holt, P.J.L Immune abnormalities associated with chronic mucocutaneous candidiasis. Cell. Immunol. 6: of Micro- Requests for reprints should be addressed to Dr. Kunisuke Himeno, Department biology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.