In vivo cell mediated immune response to M. tuberculosis and M. salmoniphilum in rainbow trout (Salmo gairdneri)

DE~/ELOPM]~NT~LLAND COP[PAI~%TIVEIMMUNOLOGY, Vol. 5, pp. 75-83, 1981 0145-305X/81/010075-09502.00/0 Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

IN VIVO CELL MEDIATED IMMUNERESPONSE TO w, TUBERCULOSIS AND M. SA.~MON/'P~Z/L~7,~ IN RAINBOW TROUT (SAZaO aAIRD~'gRI)l

James M. Bartos and Cynthia V. Sommer Center for Great Lakes Studies, University of Wisconsin-Milwaukee Department of Zoology/Microbiology, University of Wisconsin-Milwaukee Milwaukee Wisconsin 53201

ABSTRACT The cell mediated immune response to Mycobacteri~m tuberculosis and Mycobaoterium salmoniphilwn was studied in rainbow trout (SaZmo g a i r ~ e ~ ) by delayed hypersensitivity skin reactions, thymus imprint analysis and a modified skin window assay. Test fish immunized with complete Freund's adjuvant containing H. ~herc~osie were compared to control fish which received either incomplete Freund's adjuvant or phosphate buffered saline. Skin testing produced typical delayed hypersensitivity reactions with a significant difference between the test and control fish. Cross r e a c t i v i t y was produced when sensitized'fish were tested with M. s a ~ n i p h i l ~ . Imprints of thymus tissue from sensitized t r o u t showed a significant s h i f t in cell populations. Skin window studies supported the presence of cell mediated immunity by demonstrating the presence of activated macrophages at the dermal layer. INTRODUCTION The existence of d i s t i n c t T lymphocyte or T-like cell functions in fishes has been suggested by the demonstration of hapten-carrier effects (1, 2), a l l o g r a f t rejection (3), mttogen responses (4,5), and mixed leukocyte reactions (6). The extent of evolutionary development of the~e T cell functio.ns compared to the well characterized mammalian system is clouded, however, because of data showing a lack of IgG-11ke antibodies following primary and secondary antigenic responses (1,7), the existence of tmmunoglobulin-ltke molecules on nearly all lymphocytes (8), and variation in temperature and response optimum following mitogen and cell stimulation (4,10). The limited

lcontribution No. lg7. Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 5320l 75

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number of studies on the vast number of species make i t d i f f i c u l t at the present time to generalize about the phylogenetic relationship of T-like lymphocytes in fish. The question of T lymphocyte function was examined in this study by measuring the in vivo response of rainbow trout (SaZmo gairdneri) to the T cell antigen Myoobaoterium tuberculosis and a related natural fish pathogen Myoobaeterium salmoniphilum. The in vivo approach limited variations in temperature and physiological condition which are a problem in in vitro studies. Delayed hypersensitivity skin (DHS) reactions, thymus imprints from stimulated tissue and a modified Rebuck skin window assay ( l l ) for leukocyte migration were used to assess cell mediated immunity in the rainbow trout. Fish sensitized to M. tuberculosis produced t y p i c a l DHS reactions to M. t-ubereulosis and M. salmoniphilu~n, a s h i f t in cell populations in the thymus a f t e r antigen s t i m u l a t i o n , and the presence of activated macrophages in the dermis following exposure to M. salmoniphil~n. These studies support the existence of T - l i k e cell functions in rainbow t r o u t . MATERIAL AND METHODS Experimental Animals One to two year old rainbow trout (Salmo gairdneri) weighing 200-550 gm were used in all experiments. These fish, obtained from the Wisconsin Department of Natural Resources were maintained at lO-12°C on Enriched Trout Grower pellets (Glencoe Mills Inc., Glencoe, Minn.). Immunization Test fish received two peritoneal injections at 3-4 week intervals of 0.2 ml of a l : l mixture of complete Freund's adjuvant containing killed M. tubereulosis H37 (l mg/ml) (Difco, Detroit) and phosphate buffered saline, O.15 M, pH 7.3 (PBS 7.3). Control fish were immunized with either incomplete Freund's adjuvant (I~A) or PBS 7.3. Tricaine methanesulfonate (MS-222, Crescent Research Chemicals, Inc., Scottsdale, Arizona) at a concentration of lO0 mg per l i t e r was used to anesthetize the fish during all injection and surgical procedures. The protocol for immunization and testing is shown in Figure I. Delayed Hypersensitivity Skin Reactions On week 14, test and control fish were injected subcutaneously with O.l ml of each of the following preparations suspended in PBS 7.3: l . ) Heatkilled M. tuberculosis H37 (5 mg/O.l ml ), 2.) Formalin-killed M. salmoniphilum (5 mg/O.l ml), and 3.) Phosphate buffered saline, pH 7.3. Skin test reactions were read for induration and swelling at 24, 48, and 120 hours. The results from the test and control fish were evaluated s t a t i s t i c a l l y by the student T-test. Thymus Test Three weeks after secondary immunization, test and control fish were challenged with purified protein derivative (PPD) from M. tuberculosis H37 (Parke Davis and Co., Detroit). Using a 25 gauge needle, O.l ml of PPD (5 tuberculin units/O.l ml) was injected into the thymus which in trout is located under the operculum and above the g i l l arch. Seventy-two hours after challenge with PPD, imprints were made from excised thymus tissue. The

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Test- CFA

77

Control - I FA Four

Reimmunized

Control -PBS Weeks

Reimmunized Three

I

Reimmunized Weeks

Thymus Imprints 72 hrs After Challenge with PPD

I

Seven Weeks v Skin Tests

M. tuberculosis, M. salmoniphilum,

Skin Window PBS

Reactions read after 24, 48, and 120 hrs FIG I .

M. 8almoniphilum Removed 48 hrs after stimulation

Immunization and Testing Protocol

slides were stained with Wright's stain and a total of 200 cells were counted from each imprint in a double blind study. The cell populations were c l a s s i fied as small or large lymphocytes, blast or mitotic c e l l s . The student Ttest was used to determine the significance of mean differences between test and controls. Skin Window Technique Silicone elastomer polymerized on a mylar sheet served as the o p t i c a l l y clear coverslip for the modified Rebuck Skin Window Assay ( l l ) . The film was prepared by mixing ten parts of Sylgard 184 silicone elastomer with one part curing agent (Dow Corninq, Midland, Michigan). Entrapped a i r was removed by suctioning the l i q u i d for 15-20 minutes. The mixed silicone polymer was allowed to flow onto the surface of a O.l mm thick mylar sheet and cured for 7-I0 days at room temperature in a dust free environment. The resulting film which was 0.6 mm thick was cut into 1.5 x 1.5 cm squares. In the surgical procedure, the dermis layer of the caudal peduncle region of an anesthetized trout was exposed by gently scrapping away the scales and epidermis with a s t e r i l e BP #22 blade. A volume of 0.05 ml of a heat-killed suspension of M. salmoniphilum (l mg/O.05 ml) was dropped onto the 4 cm square area of exposed dermal tissue. The silicone film with the mylar backing facing outward, was placed onto the exposed tissue. The film was i n i t i a l l y attached to a 3.0 cm wide Gooch rubber tubing with cyanoacrylic adhesive. The rubber tubing was then secured to the fish with two sutures placed through the tubing at sites above and below the film. The Gooch tubing was f i n a l l y attached to i t s e l f with adhesive to form a rubber band around the f i s h . At 48 hours, the silicone film was removed, transferred to a microscope s l i d e , and stained with Wright°s stain. Cell counts of the attached leukocytes were made and macrophages were differentiated into activated and non-activated c e l l s . Activated macrophages were classified as cells with active membranes, pseudopods, and vacuoles.

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RESULTS Delayed Hypersensitivit£ Skin Test The typical induration and swelling characteristics of delayed hypers e n s i t i v i t y (DHS) skin reactions were noted at the site of injection of both M. tuberculosis and M. salmoniphil~n but not with the PBS 7.3 solution. I t was found necessary to challenge the fish with particulate bacterial suspensions rather than soluble extracts to produce DHS reactions. Attempts to produce skin reactions in sensitized fish with soluble PPD (5 T.U./O.I ml) resulted in no response. Test fish produced s i g n i f i c a n t l y stronger reactions to M. tuberculosis than either the incomplete Freund's adjuvant or PBS controls at 24, 48, and 12O hours (Table l ) . The optimum time for reading the DHS reaction was determined to be 48 hours. Cross r e a c t i v i t y was seen in test fish challenged with M. salmoniphilu~n. At 48 hours, there was a significant difference between the strength of the reaction in CFA-sensitized fish to M. salmoniphilzan when compared to both control groups. The s p e c i f i c i t y of the DHS reactions was evident, however, in that M. tuber~losis produced s i g n i f i c a n t l y greater reactions in test fish than did M. salmoniphil~n. Significant differences were not observed between the reaction to M. tuberculosis and the reaction to M. salmoniphilum in IFA controls. T_hymus Test The results of thymus imprints made from test and control fish 72 hours after challenge with PPD are seen in Table 2. A s h i f t in the population of lymphocytes was observed in the CFA-sensitized fish when compared to the IFA and PBS controls. Differential cell counts showed that the test fish had s i g n i f i c a n t l y fewer small !ymphocytes and more large lymphocytes than either control. The thymic tissue of test fish appeared on gross examination more swollen than either of the controls. No significant difference in the number of mitotic cells was noted between the test and control fish. A small group of test and control fish retested 4 weeks later with a second injection of PPD into the regenerated thymus produced a specific memory response. An even greater s h i f t (40% of total cell count) to large lymphocytes was seen in the test fish. Skin Window Assax The f l e x i b l e , optically clear silicone elastomer film allowed adaptation of the skin window assay to fish. The major cell type observed at the dermal layer 48 hours after stimulation with M. salmoniphilu~n was the macrophage (Table 3). The PBS control fish had the greatest population of l~nnphocyte and pol~nBorphonuclear cells. Test fish showed a significant s h i f t to activated macrophages compared to the PBS control fish.

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TABLE 1 Delayed H y p e r s e n s i t i v i t y Skin Reactions To

M. tuberculosis

and M.

8almoniphilum

DHS Mean Reaction

Size (mm2±S.D.)

Experimental Group # o f Fish

24 Hours

48 Hours

Test - CFA

lO

356+ 87.7 * t

663+ 140.7 * t

463+ 94.9* t

lO

201 + 73.4**

413-+ 121.4"

352-+ 126.9"*

II

153-+ 53.8

202-+ 87.0

245-+ 45.5

II

155-+ 53.9

153-+ 49.6

209-+ 84.1

11

90+ 50.0

281+ 94.1 t

220+ 60.1 t

II

66+ 37.9

153-+ 62.8

131 + 63.9

120 Hours

(M. tuberculosis ) Test - CFA

(M. "salmoniphilum ) Control - IFA

(M. tuberculosis ) Control - IFA

(M. 8almoniphilum ) Control - PBS

(M. tuberculosis ) Control - PBS

(M. salmoniphilum)

P < O.OOl, Test-CFA compared to Control-IFA and Control-PBS by student T-test P < O.OOl, Test-CFA compared to Control-PBS by student T-test tp < 0.05, M. tuberculosis compared to M. salmoniphilum by student Ttest CFA - complete Freund's adjuvant containing IFA - incomplete Freund's adjuvant PBS - phosphate buffered saline, pH 7.3

M. tuberculoeis H37

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TABLE 2 IThymus Imprints 72 Hours Post-Challenge With PPD Experimental Group

# of Fish

Small Lymphs

Large Lymphs

Blast Cells

Mi to t i c Cel I s

Test - CFA

12

121+ 14.0"

63+ I 0 . 8 "

14+ 7.2

2+ 1.2

Control - IFA

II

159+ 8.5

27+ 9.6

13+ 6.4

l + l.O

Control - PBS I I

163-+ 9.0

27+ 7.4

8-+ 5.4

2+ 1.4

1200 c e l l s were counted from each imprint P < O.OOl, Test-CFA compared to Control-IFA and Control-PBS by student T-test PPD - p u r i f i e d protein d e r i v a t i v e TABLE 3 Percentage Of Various Leukocytes Present At The Dermal Layer In Response To

~cobaote~n salmoniphil~m Using Skin Window Technique Percentage of Cell Population+ S.D.

Experimental Group

# of Fish

Activated Macrophages

Non-Activated Macrophages

Ljnnphocytes and PMN's

Test - CFA

8

55+ l l . 4 *

35+ 5.6

lO + I I .9

Control - IFA

8

45+ 15.4

49+ 13.4

6+ 9.6

Control - PBS

lO

34+ 15.8

39+ 14.2

27+ 26.8

P < O.Ol, Test-CFA compared to Control-PBS by student T-test PMN - polymorphonuclear leukocyte DISCUSSION The hypothesis that bony f i s h have a population of lymphocytes akin to the T cell of higher vertebrates is supported by the results of our study of c e l l mediated immunity in rainbow t r o u t . The skin reaction to M. ~ubereulosi8 was a s p e c i f i c memory response with c h a r a c t e r i s t i c s s i m i l a r to the reaction of mammals. The extent of induration and swelling in the sensi-

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tized fish was shown to be s i g n i f i c a n t l y different than either the IFA or PBS controls. Even at lower temperatures of lO-12°C, the optimum time of reaction was found to be 48 hours. There was also significant induration remaining after 120 hours which is probably due to the rate of clearance of the particulate antigen used for challenge. In contrast to reactions seen in higher vertebrates, the skin reaction in trout was produced only with particulate suspensions of the microorganism and not with the standard soluble extract PPD. These results may explain the work of Hodgins (12) in which inconclusive, weak to moderate corneal reactions occurred in sensitized fish exposed to tubercul i n . The processing or binding of soluble antigen may not be as highly developed in the teleost as in the higher vertebrates. Cross r e a c t i v i t y was also observed when test fish immunized with M. tuberculosis were tested with a killed bacterial suspension of M. salmoniphilum. This is not unexpected since cross r e a c t i v i t y among Mycobacterium species is well documented in human studies (13). The type of response produced by both of these Mycobacterium cell suspensions indicates that cell mediated immunity can be produced at the dermal layer. The thymus test results showed an increase in the number of large lymphocytes compared to either IFA or PBS controls. I t has been shown that large lymphocytes and blast cells are produced followinB in vitro stimulation of small lymphocytes by mitogen or specific antigen (14). A question to raise is whether the phenomenon observed is due to a T-like cell population since PPD can act as a B cell mitogen (15). However, the lymphocyte heterogeneity studies of Etlinger et al. (5,10) showed that thymocytes from rainbow trout are selectively stimulated by the T cell mitogen concanavalin A but not by PPD or lipopolysaccharide. Also, the lack of lymphocyte activation in the thymus of both IFA and PBS controls, which were also injected with PPD, indicates that the lymphocyte activation seen in the CFA test fish i s , in fact, the result of antigenic and not mitogenic stimulation. An increased s h i f t in the number of large lymphocytes in sensitized fish stimulated twice in thymus tests further indicates the antigenic nature of the response. The possibili t y of a B cell antigen response could exist since antibody production has been shown in some teleosts (17). The type of antigen used in this study and the observation of only an occasional plasma cell makes i t more probable that we are dealing with a T cell response. The results of the s~in window test further support the existence of a T c e l l - l i k e response in rainbow trout. In our observation, macrophages were the predominant cell in the dermis at 48 hours after stimulation in all groups of fish, but the test fish showed an increase in the number of activated macrophages. The presence of activated macrophages suggest the possible release of lymphokines which is most often associated with T cell function. The presence of in v i t r o MIF responses has been demonstrated in teleosts (18). A more dramatic response may have been produced with the homologous antigen. The optimum time of reaction and the type of specific responses seen following stimulation with a T cell antigen support the existence of T cell function with anamnesis in the rainbow trout. These i n vivo approaches have minimized temperature and physiological variations which are a problem in evaluating in v i t r o responses. The presence of a significant cross reactive response to the natural pathogen M. salmoniphilum warrants further studies into protective cell mediated immune responses to infectious microbes.

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REFERENCES I.

YOCUM,D., CUCHENS, M., and CLEM, L.W. The hapten-carrier effect in teleost fish. J. Immunol. l l 4 , 925, 1975.

.

RUBEN, L.N., WARR, G.W., DECKER, J.M., and MARCHALONIS, J.J. Phylogenetic origins of immune recognition: L3nnphoid heterogeneity and the hapten/carr i e r effect in the goldfish, Carassius auratus. Cell. Immunol. 31, 266, 1977.

.

HILDEMANN, W.H. Transplantation immunity in fishes: agnatha, chondrichthyes and osteichthyes. Transpl. Proc. 2, 253, 1970.

.

CUCHENS, M.A. and CLEM, L.W. Phylogeny of 13nnphocyte heterogeneity. Differential effects of temperature on fish T-like and B-like cells. Cell. Immunol. 34, 219, 1977.

.

ETLINGER, H.M., HODGINS, H.O., and CHILLER, J.M. Evolution of the lymphoid system. I l l . Morphological and functional consequences of mitogenic stimulation of rainbow trout lymphocytes. Develop. Comp. Immunol. 2, 263, 1978.

.

ETLINGER, H.M., HODGINS, H.O., and CHILLER, J.M. Properties of rainbow trout lymphocytes, mitogenic stimulation, surface Ig and mixed leukocyte reactions. Fed. Proc. 34, 966, 1975.

7.

TRUMP, G.N. Goldfish immunoglobulins and antibodies to bovine serum albumin. J. Immunol. I04, 1267, 1970.

.

9.

II.

WARR, G.W., DELUCA, D., and MARCHALONIS, J.J. Phylogenetic orgins of immune recognition: Lymphocyte surface immunoglobulins in the goldfish Cara88ius auratus. Proc. Nat'l. Acad. Sci. USA 73, 2476, 1976. ETLINGER, H.M., HODGINS, H.O., and CHILLER, J.M. Characterization of lymphocytes in a primitive teleost, Salmo gairdneri. In: Phylogeny of Th~nnus and Bone Marrow. R.K. Wright and E.L. Cooper (Ed.) New York and Amsterdam, North-Holland, 1976, p. 83.

lO.

ETLINGER, H.M., HODGINS, H.O., and CHILLER, J.M. Evolution of the lymphoid system. I. Evidence for 13nnphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. J. of Immunol. If6, 1547, 1976.

II.

REBUCK, G.J. and CROWLEY, J.H. A method of studying leukocytic functions in vivo. Ann. N.Y. Acad. Sci. 59, 757, 1955.

12.

HODGINS, H.O. Studies of mechanisms of immunity in rainbow trout (Salmo gairdneri). Ph.D. Thesis, Univ. of Washington, USA, 1966.

13.

WILSON, G.S. and MILES, A. In: Topley and Wilson's Principles of Bacteriology, Virology, and Immunology. Baltimore: The Williams and Wilkins Co. 1975.

14.

BLOOM,B.R. and GLADE, P. In Vit~ Methods in Cell-Mediated Immunity. New York: Academic Press, 1970.

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15.

ANDERSSON, J., MOLLER, G., and SJOBERG, O. Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4, 381, 1972.

16.

SULTZER, B.M. and NILSSON, B.S. PPD-tuberculin - a B cell mitogen. Nature (New Biol.) 240, 198, 1972.

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ORTIZ-MUNIZ, G. and SIGEL, M.M. Antibody synthesis in lymphoid organs of two marine teleosts. J. Retriculo. Soc. 9, 42, 1971.

18.

MC KINNEY, E.C., ORTIZ, G., LEE, J.C., SIGEL, M.M., LOPEZ, D.M., EPSTEIN, R.S. and MC LEOD, T.F. Lymphocytes of fish: Multipotential or specialized? In: Phylogeny of Thymus and Bone Marrow. R.K. Wright and E.L. Cooper (Ed.) New York and Amsterdam, North-Holland, 1976, p. 73.