CELLULARIMMUNOLOGY17, 335-341 (1975)
Capillary Tube Leukocyte Migration Inhibition as a Correlate of Cell-Mediated Immunity in the Chicken 1 MILAN S. VLAOVIC,2 GERALD M. BUENING, AND RAYMOND W . LOAN
Department of Veterinary Microbiology, University of Missouri, Columbia, Missouri 65201 Received rune 21, 1974 The migration of peripheral blood leukocytes from white leghorn roosters sensitized to mycobacteria was significantly inhibited in the presence of PPD. Positive leukocyte migration inhibition tests (LMIT) preceded positive wattle tests in 11 of 34 chickens. In 1 of 42 chickens a positive wattle test preceded positive LMIT. Repeated injections of PPD alone in some chickens resulted in 20% or more inhibition of migration of leukocytes in the presence of PPD ( P < 0'.05) even though these chickens had negative dermal reactions. Sera from these chickens contained cytophilie antibodies which rendered normal peripheral blood leukoeytes sensitive to inhibition by PPD. Serum from one chicken sensitized to mycobacteria also contained cytophilic antibody. Cytophilic antibody was not found in autologous or homologous sera from control chickens. Rabbit anti-chicken IgG abolished leukocyte migration inhibition due to cytophilic antibodies, but had little or no effect on migration inhibition of sensitized cells. The results of these tests indicate that LMIT in chickens are reproducible and parallel that of the human. The addition of rabbit anti-chicken IgG to the LMIT allowed differentiation of leukocyte migration inhibition due to cell-mediated immunity from that due to cytophilic antibody. INTRODUCTION Techniques for the in vitro measurement of cell-mediated immunity ( C M I ) in the chicken have been developed using small spleen fragments (1, 2), individual spleen cells (3, 4), chicken peritoneal cells (5), and chicken peripheral blood leukocytes (6). T h e results of these tests were in general agreement with delayed skin test reactions. In vitro tests for C M I were not affected by bursectomy (2, 5), but were thymus dependent (2). Highly purified antigen fractions used in the in vitro tests gave better correlation with skin tests than the corresponding crude antigen preparations (6). Sjogren and Jonsson (7) investigated C M I against tumor specific transplantation antigens in Rous sarcoma bearing chickens. Using the colony inhibition technique (8), these investigators observed significant inhibition of Rous t u m o r cell colony formation by t h y m u s cells from 87.5% of Rous sarcoma bearing chickens. T h e y did not observe thymus cell inhibition of colony formation by cells from other t u m o r types in the above tests. 1 This investigation was supported by N I H Grant Numbers 11309 and 13134 from the National Cancer Institute. 2 Present address : Frederick Cancer Research Center, Frederick, Maryland 21701. 335 Copyright~ 1975by AcademicPress, Inc. All rights of reproductionin any form reserved.
336
VLAOVIC, BUENING AND LOAN
This study was designed to adapt L M I T for use with avian lymphoid cells from peripheral blood, to elucidate the onset of CMI as measured by LMIT, and to investigate the possible effect of cytophilic antibody on L M I T in the avian species. MATERIALS AND METHODS
Immunization of Experimental Animals. Six-month-old white leghorn roosters were injected in each footpad with 0.5 ml Freund's complete adjuvant containing 5 mg of H 37 Ra Mycobcwterium tuberculosis. These were referred to as myeobacteria sensitized chickens and leukocytes from these chickens were sensitized leukocytes. Cytophilic antibodies were induced by four weekly intradermal wattle injections with 10 #g of purified protein derivative (PPD) 3 in 0.1 ml of Puck's a PPD--(Parke Davis, Inc.)--48.2% tuberculoproteln. saline G. These were referred to as the tuberculin immune chickens and leukocytes from these chickens with antibody attached were referred to as cytophilic antibody armed leukocytes. Uninjected chickens were referred to as control chickens and leukocytes from these chickens were referred to as normal leukocytes. Culture Medium. Roswell Park Memorial Institute (RPMI) 1640 medium was supplemented with 10% inactivated fetal calf serum (pretested to support leukocyte migration), 100 ~g/ml of streptomycin, and 100 units/ml of penicillin. Leukocyte Migration Inhibition Tests. Ten milliliters of blood with a final concentration of 10 units of heparin/ml was obtained from mycobacteria sensitized and control chickens. The blood was centrifuged at 25g for 15 rain within 10 rain of collection. Leukocyte rich plasma was aspirated. The leukocytes were washed three times by centrifugation at 150g for 10 rain in culture medium. After the third washing, the leukocyte suspension from each chicken was divided into two equal aliqnots, transferred to 12 × 75 mm tubes and centrifuged at 1509 for 10 rain. The leukocyte pellet from one tube was resuspended in culture medium at a final concentration of 5 × l0 T to 1 × 10s cells/ml (control culture). The leukocyte pellet in the second tube was resuspended to the same concentrations in medium containing 100 ~g/ml of PPD (test culture). Cell suspensions were placed in coagulation capillary tubes and centrifuged at 500g for 5 rain. The tubes were cut at the fluid-cell interface and the portion containing the cell pellet was placed on a plastic cover slip (for tissue culture) in a Mackaness type migration chamber. The Capillary tubes were held in place by means of a small amount of si!icone lubricant and the top plastic cover slip was fixed in place with the same lubricant. The test culture migration chamber was filled with approximately 1.0 ml of culture medium containing 100 ~g of PPD, while the corresponding control culture chamber was filled with culture alone. Migration chambers were incubated at 37°C for 18 hr. The area of migration was projected on bond paper (ll-fold magnification), traced, cut out, and weighed with an analytical balance. Calculation. The percentage of migration inhibition was calculated according to the following formula : 100
-
Area of migration (weight) in test chamber Area of migration (weight) in control chamber
X
100
-- % migration inhibition. Analysis of variance data from this study confirmed previously reported findings (9) which indicated that inhibition of 20% was statistically significant (P < 0.05).
LEUKOCYTE MIGRATION INHIBITION
IN THE CHICKEN
337
Detection of Cytophilic Antibody. Sera from mycobacteria sensitized, tuberculin immune, and control chickens were tested for the presence of cytophilic antibodies using the technique of Amos et al. (10). Specifically, normal peripheral blood leukocytes were washed three times by centrifugation in medium at 150g for 10 rain to remove normal plasma proteins. The leukocytes were resuspended in 4.0 ml of a 1:10 dilution of appropriate sera from mycobacteria sensitized, tuberculin immune, or control chickens. After 1 hr incubation at room femperature, the cells were washed three times in culture medium, by centrifuging at 150g for 10 min at 4°C. The leukocyte suspension was divided into two equal aliquots and L M I T were performed. Autologous and homologous sera were used in negative control tests. Effect of Rabbit Anti-Chicken IgG on L M I T . This effect was measured by a modification of the method of Rajapakse et al. (11). Suspensions of peripheral blood leukocytes previously incubated with appropriate sera and subsequently washed three times were incubated at 4°C for 30 rain with 0.5 ml of a 1:5 dilution of rabbit anti-chicken IgG obtained from Cappel Laboratories, Inc., Downingtown, Pa. The leukocytes were washed three additional times in culture medium and used in LMIT. Skin tests. Skin testing was performed, after blood samples were obtained, by the intradermal injection of 10 ~g of P P D in 0.1 ml of Puck's saline G in one wattle. As a control 0.1 ml of Puck's saline G was injected into the opposite wattle. The wattles were alternatively injected with P P D and Puck's saline G to compensate for possible errors due to induration. Degree of swelling was measured 24 hr postinjection with the aid of calipers. The skin reactions were graded as positive if the wattle thickness had increased at least 100% compared to the opposite saline injected wattle control. This was comparable to strong positive reactions as reported by Theis and Thorbecke (12). RESULTS During the sequential study of the onset of CMI to M. tuberculosis as measured by LMIT, a total of 62 samples from 10 control chickens and 58 samples from 10 immunized chickens were tested. Throughout the entire study, the mean percentage of migration inhibition from control chickens was negative; i.e., the migration areas were larger in the presence of P P D than in the absence of P P D ( - 9 . 2 9 to -44.91% migration inhibition). For the 10 wk after immunization, the mean percentage of migration inhibition from immunized birds was -14.23% to 65.00% (Fig. 1). During the first 4 wk postinjection, 11 leukocyte samples from 5 chickens with negative wattle tests had positive L M I T ; however, 27 of 28 tests from 10 birds with positive wattle tests had significant LMIT. Ten chickens not sensitized to myeobacteria were P P D wattle tested at weekly intervals during the first 4 wk of study. In the process of this testing, these chickens apparently became immune to PPD. One of these chickens had 25.5% migration inhibition when tested by L M I T during the second week. During the first 8 wk of the study, 9 of 54 L M I T from these chickens had 20% or more migration inhibition (Table 1). None of these chickens had positive intradermal wattle tests. When tested for the presence of cytophilic antibodies 10 wk after the initial P P D
338
VLAOVIC, BUENING AND LOAN
1oo 90 80 o
70
60 5O 4o
~.
30
./ / WEEKSPOSTINJECTION
Fro. 1. Comparison of leukocyte migration inhibition tests ( 0 O ) with wattle tests (X X ) for delayed hypersensitivity in mycobacterial sensitized chickens.
injection, 7 of 10 sera from these PPD immune chickens had cytophilic antibodies. Only one chicken sensitized to mycobacteria had cytophilic antibody (Table 2). To differentiate positive LMIT due to CMI from those due to cytophilic antibodies, an additional treatment with anti-chicken IgG was introduced (Table 3). Normal chicken peripheral blood leukocytes with passively acquired cytophilic antibody were significantly inhibited in their migration in the presence of PPD. After treatment with rabbit anti-chicken IgG, migration inhibition of peripheral blood leukocytes with cytophilic antibodies was reduced to insignificant levels. Migration inhibition of peripheral blood leukocytes from chickens immunized with Freund's complete adjuvant were not affected. DISCUSSION Delayed hypersensitivity in the chicken as measured by wattle tests (positive test = 100~ increase in thickness) was first detected 2 wk after injection of TABLE 1 INHIBITION OF MIGRATION OF LEUKOCYTES FROM TUBERCULIN IMMUNE CHICKENS a IN THE PRESENCE OF P P D
Week of test
Number with significant LM ITb/number tested
1~ 2c 3c
0/7 1/10 2/10
4c
1/8
6 8
3/10 2/9
a Chickens were not sensitized to mycobacteria. b Significant L M I T = 20% or more migration inhibition. c Wattle injections with 10 ~g P P D in 0.1 ml of Puck's saline.
LEUKOCYTE MIGRATION I N H I B I T I O N I N T H E C H I C K E N
339
TABLE 2 EFFECT OF SERA FROM MYCOBACTERIA SENSITIZED AND PPD IMMUNE CHICKENS ON THE LEUKOCYTE MIGRATION INHIBITION TEST (LMIT)
Serum source
Number of sera with significant LMIT*/number of sera tested
Normal chicken (Autologous)a Normal chicken (Homologous)~ PPD immunechickenb Mycobacteria sensitizedchicken
0/9 0/9 7/10 1/10
a Sera from normal chickens. bSera tested at 10 wk after initial PPD injection. c SignificantLMIT = 20% or more migrationinhibition. Freund's complete adjuvant. Four weeks after injection, 50% of the chickens had developed delayed hypersensitivity. All chickens became hypersensitive by the sixth postinjection week (Fig. 1). Footpad inoculation in the mycobacteria sensitized chickens resulted in an inflammatory response that was initially observed at 1 wk postinjection. This reaction was prominent during the following 3 wk and then subsided. Failure to obtain significant inhibition of migration of peripheral blood leukocytes in 18 chickens during the first 4 wk postinjection could be partially attributed to the accumulation of sensitized leukocytes in the area of inflammation. Similar effects were observed when wattle testing preceded the lymphoid cell harvest by 18-24 hr (4). In studies of CMI, Fauser et al. (6) replaced capillary tubes in the migration inhibition test with drops of cell suspension approximately 4 mm in diameter placed in plastic Petri dishes. Using this technique, valuable data on the role of CMI and humoral antibodies in Marek's disease were obtained. In experiments reported here, some peripheral blood leukocytes (up to 30%) from the tuberculin immune group (not sensitized to mycobacteria) were inhibited in their migration in the presence of PPD (Table 2). Since none of these birds had positive wattle tests at any time during the experiment, the presence of cytophilic antibodies was suspected and later confirmed. Ten weeks after initial wattle testing, 70% of the serum samples from these chickens contained sufficient quantities of eytophilic antibodies to cause normal chicken peripheral blood leukocytes to be inhibited in the presence of PPD (Table 3). Zwilling et al. (3) were unable to detect cytophilic antibody armed cells following the intravenous injection of chickens with bovine gamma globulin. However, in their study four to six sera from chickens with precipitating antibodies to PPD inhibited normal cell migration in the presence of PPD. Immunization schedules that usually lead to the appearance of CMI can produce cytophilic antibodies which are capable of inhibiting the migration of normal macrophages (10). Sera from human patients allergic to penicillin rendered leukocytes from nonallergic individuals susceptible to inhibition when exposed to penicillin antigen (13). The substance involved in this reaction was identified as an antibody. Migration inhibition due to CMI or cytophilic antibodies was indistinguishable by L M I T alone. Treatment with rabbit anti-chicken IgG differentiated these two types of migration inhibition. Rabbit anti-chicken IgG rendered migration inhibition
340
:
VLAOVIC~BUENING AND LOAN TABLE 3
EFFECT OF RABBIT ANTI-CHICKEN IgG ON TKE MIGRATION OF CHICKEN PERIPHERAL BLOOD LEUKOCYTES Reaction mixture
Percentage of migration inhibition
LM IT pos./ no. tested
Normal leukocytes + PPD~ Normal leukocytes
--0.7, --9.5
0/2
Normal leukocytes + cyt. Ab b + PPD Normal leukocytes + cyt. Ab
35.5, 32.5, 34.1
3/3
Normal leukocytes + NChS * + PPD Normal leukocytes + NChS
-21.6, --2.2
0/2
Normal leukocytes + cyt. Ab + Anti IgG d + PPD Normal leukocytes + cyt. Ab + Anti IgG
--6.4, --2.1
0/2
Normal leukocytes + cyt. Ab + NRS ~ + PPD Normal leukocytes + Ab + NRS
29.8, 25.6
2/2
Normal leukocytes + NChS + NRS + PPD Normal leukocytes + NChS + NRS
--2.7, --9.3
0/2
Cyt. Ab armed leukocyteJ + PPD Cyt. Ab armed leukocytes
31.0, 25.2, 24.5
3/3
Cyt. Ab armed leukocytes + Anti IgG + PPD Cyt. Ab armed leukocytes q- Anti IgG
8.0, 3.7, --9.6
0/3
Cyt. Ab armed leukocytes q- NRS -t- PPD Cyt. Ab armed leukocytes + NRS
23.2, 24.7, 23
3/3
Sensitizedg leukocytes + PPD Sensitized leukocytes
21.6, 25.0, 24.7, 23.8, 24.3, 10.0, 8.0
5/7
Sensitized leukocytes + Anti IgG + PPD Sensitized leukocytes + Anti IgG
24.3, 24.4, 24.3, 23.1, 22.7, 10.2, 9.1
5/7
Sensitized leukocytes + NRS + PPD Sensitized leukocytes + NRS
35.9, 32.6, 34.2, 32.7, 29.1, 21.2, 20.4
7/7
PPD = purified protein derivative of tuberculin. b Cyt. Ab = Cytophilie antibody from PPD tested birds with significant leukocyte migration inhibition test (LMIT). c NChS = normal chicken serum. d Anti IgG = rabbit anti-chicken IgG. e NRS = normal rabbit serum. s Cyt. Ab armed leukocytes = leukocytes from PPD immune chickens with significant LMIT. g Sensitized leukocytes = leukocytes from chickens injected with Freund's complete adjuvant. of cytophilic a n t i b o d y - p r e t r e a t e d n o r m a l p e r i p h e r a l blood leukocytes insignificant, and abolished the m i g r a t i o n inhibition of cytophilic antibody a r m e d leukocytes. I t did not affect m i g r a t i o n inhibition due to C M I ( T a b l e 4 ) . T h i s is further evidence that chicken " T " lymphocytes do not c a r r y significant a m o u n t s of surface i m m u n o globulin d e t e r m i n a n t s ( 1 4 ) , and are not affected by rabbit anti-chicken I g G . REFERENCES 1. Morita, C., and Soekawa, N., Poultry Sci., 50, 1503-1505, 1971. 2. Morita, C., and Soekawa, M., Poultry Scl. 51, 1133-1136, 1972.
LEUKOCYTE MIGRATION INHIBITION IN THE CHICKEN 3. 4. 5. 6.
341
Zwilling, B. S., Barrett, J. T., and Breitenbach, R. P., Cell. ImmunoL 4, 20-28, 1972. @ranlund, D. J., Buening, G. M., and Loan, R. W., Cell. Immunol. 11, 99-107, 1974. Warner, N. L., Ovary, Z., and Kantor, F. S., Int. Arch. Allergy 40, 719-728, 1971. Fauser, I. S., Purchase, H. G., Long, P. A., Velicer, L. F., Mallmann, V. t-I., Fauser, H. T., and Winegar, G. D., Avian Pathol. 2, 55-61, 1973. 7. Sjogren, H. O., and Jonsson, N., Cancer Res. 30, 2434-2437, 1970. 8. Hellstrom, I., Intern. J. Cancer 2, 65-68, 1967. 9. Pick, E., and Turk, J. L., Clin. Exp. Immunol. 10, 1-23, 1972. 10. Amos, H. E., Gurner, B. W., Olds, R. J., and Coombs, R. R. A., Int. Arch. Allergy 32, 496-505, 1967. I1. Raj~/pakse, D. A., Papamichail, M., and Holborow, E. J., Nature New Biol. 245, 155-157, 1973. 12. Theis, G. A., and Thorbecke, G. J., )'. Immunol. 110, 91-97, 1973. 13. Ortiz-Ortiz, L., Zamacona, G., Garmilla, C., and Arellano~ M. T., J. Immunol. 113, 993997, 1974. 14. Alb'ni, B., and Wick, G., Int. Arch. Allergy 44, 804-822, 1973.
Capillary Tube Leukocyte Migration Inhibition as a Correlate of Cell-Mediated Immunity in the Chicken 1 MILAN S. VLAOVIC,2 GERALD M. BUENING, AND RAYMOND W . LOAN
Department of Veterinary Microbiology, University of Missouri, Columbia, Missouri 65201 Received rune 21, 1974 The migration of peripheral blood leukocytes from white leghorn roosters sensitized to mycobacteria was significantly inhibited in the presence of PPD. Positive leukocyte migration inhibition tests (LMIT) preceded positive wattle tests in 11 of 34 chickens. In 1 of 42 chickens a positive wattle test preceded positive LMIT. Repeated injections of PPD alone in some chickens resulted in 20% or more inhibition of migration of leukocytes in the presence of PPD ( P < 0'.05) even though these chickens had negative dermal reactions. Sera from these chickens contained cytophilie antibodies which rendered normal peripheral blood leukoeytes sensitive to inhibition by PPD. Serum from one chicken sensitized to mycobacteria also contained cytophilic antibody. Cytophilic antibody was not found in autologous or homologous sera from control chickens. Rabbit anti-chicken IgG abolished leukocyte migration inhibition due to cytophilic antibodies, but had little or no effect on migration inhibition of sensitized cells. The results of these tests indicate that LMIT in chickens are reproducible and parallel that of the human. The addition of rabbit anti-chicken IgG to the LMIT allowed differentiation of leukocyte migration inhibition due to cell-mediated immunity from that due to cytophilic antibody. INTRODUCTION Techniques for the in vitro measurement of cell-mediated immunity ( C M I ) in the chicken have been developed using small spleen fragments (1, 2), individual spleen cells (3, 4), chicken peritoneal cells (5), and chicken peripheral blood leukocytes (6). T h e results of these tests were in general agreement with delayed skin test reactions. In vitro tests for C M I were not affected by bursectomy (2, 5), but were thymus dependent (2). Highly purified antigen fractions used in the in vitro tests gave better correlation with skin tests than the corresponding crude antigen preparations (6). Sjogren and Jonsson (7) investigated C M I against tumor specific transplantation antigens in Rous sarcoma bearing chickens. Using the colony inhibition technique (8), these investigators observed significant inhibition of Rous t u m o r cell colony formation by t h y m u s cells from 87.5% of Rous sarcoma bearing chickens. T h e y did not observe thymus cell inhibition of colony formation by cells from other t u m o r types in the above tests. 1 This investigation was supported by N I H Grant Numbers 11309 and 13134 from the National Cancer Institute. 2 Present address : Frederick Cancer Research Center, Frederick, Maryland 21701. 335 Copyright~ 1975by AcademicPress, Inc. All rights of reproductionin any form reserved.
336
VLAOVIC, BUENING AND LOAN
This study was designed to adapt L M I T for use with avian lymphoid cells from peripheral blood, to elucidate the onset of CMI as measured by LMIT, and to investigate the possible effect of cytophilic antibody on L M I T in the avian species. MATERIALS AND METHODS
Immunization of Experimental Animals. Six-month-old white leghorn roosters were injected in each footpad with 0.5 ml Freund's complete adjuvant containing 5 mg of H 37 Ra Mycobcwterium tuberculosis. These were referred to as myeobacteria sensitized chickens and leukocytes from these chickens were sensitized leukocytes. Cytophilic antibodies were induced by four weekly intradermal wattle injections with 10 #g of purified protein derivative (PPD) 3 in 0.1 ml of Puck's a PPD--(Parke Davis, Inc.)--48.2% tuberculoproteln. saline G. These were referred to as the tuberculin immune chickens and leukocytes from these chickens with antibody attached were referred to as cytophilic antibody armed leukocytes. Uninjected chickens were referred to as control chickens and leukocytes from these chickens were referred to as normal leukocytes. Culture Medium. Roswell Park Memorial Institute (RPMI) 1640 medium was supplemented with 10% inactivated fetal calf serum (pretested to support leukocyte migration), 100 ~g/ml of streptomycin, and 100 units/ml of penicillin. Leukocyte Migration Inhibition Tests. Ten milliliters of blood with a final concentration of 10 units of heparin/ml was obtained from mycobacteria sensitized and control chickens. The blood was centrifuged at 25g for 15 rain within 10 rain of collection. Leukocyte rich plasma was aspirated. The leukocytes were washed three times by centrifugation at 150g for 10 rain in culture medium. After the third washing, the leukocyte suspension from each chicken was divided into two equal aliqnots, transferred to 12 × 75 mm tubes and centrifuged at 1509 for 10 rain. The leukocyte pellet from one tube was resuspended in culture medium at a final concentration of 5 × l0 T to 1 × 10s cells/ml (control culture). The leukocyte pellet in the second tube was resuspended to the same concentrations in medium containing 100 ~g/ml of PPD (test culture). Cell suspensions were placed in coagulation capillary tubes and centrifuged at 500g for 5 rain. The tubes were cut at the fluid-cell interface and the portion containing the cell pellet was placed on a plastic cover slip (for tissue culture) in a Mackaness type migration chamber. The Capillary tubes were held in place by means of a small amount of si!icone lubricant and the top plastic cover slip was fixed in place with the same lubricant. The test culture migration chamber was filled with approximately 1.0 ml of culture medium containing 100 ~g of PPD, while the corresponding control culture chamber was filled with culture alone. Migration chambers were incubated at 37°C for 18 hr. The area of migration was projected on bond paper (ll-fold magnification), traced, cut out, and weighed with an analytical balance. Calculation. The percentage of migration inhibition was calculated according to the following formula : 100
-
Area of migration (weight) in test chamber Area of migration (weight) in control chamber
X
100
-- % migration inhibition. Analysis of variance data from this study confirmed previously reported findings (9) which indicated that inhibition of 20% was statistically significant (P < 0.05).
LEUKOCYTE MIGRATION INHIBITION
IN THE CHICKEN
337
Detection of Cytophilic Antibody. Sera from mycobacteria sensitized, tuberculin immune, and control chickens were tested for the presence of cytophilic antibodies using the technique of Amos et al. (10). Specifically, normal peripheral blood leukocytes were washed three times by centrifugation in medium at 150g for 10 rain to remove normal plasma proteins. The leukocytes were resuspended in 4.0 ml of a 1:10 dilution of appropriate sera from mycobacteria sensitized, tuberculin immune, or control chickens. After 1 hr incubation at room femperature, the cells were washed three times in culture medium, by centrifuging at 150g for 10 min at 4°C. The leukocyte suspension was divided into two equal aliquots and L M I T were performed. Autologous and homologous sera were used in negative control tests. Effect of Rabbit Anti-Chicken IgG on L M I T . This effect was measured by a modification of the method of Rajapakse et al. (11). Suspensions of peripheral blood leukocytes previously incubated with appropriate sera and subsequently washed three times were incubated at 4°C for 30 rain with 0.5 ml of a 1:5 dilution of rabbit anti-chicken IgG obtained from Cappel Laboratories, Inc., Downingtown, Pa. The leukocytes were washed three additional times in culture medium and used in LMIT. Skin tests. Skin testing was performed, after blood samples were obtained, by the intradermal injection of 10 ~g of P P D in 0.1 ml of Puck's saline G in one wattle. As a control 0.1 ml of Puck's saline G was injected into the opposite wattle. The wattles were alternatively injected with P P D and Puck's saline G to compensate for possible errors due to induration. Degree of swelling was measured 24 hr postinjection with the aid of calipers. The skin reactions were graded as positive if the wattle thickness had increased at least 100% compared to the opposite saline injected wattle control. This was comparable to strong positive reactions as reported by Theis and Thorbecke (12). RESULTS During the sequential study of the onset of CMI to M. tuberculosis as measured by LMIT, a total of 62 samples from 10 control chickens and 58 samples from 10 immunized chickens were tested. Throughout the entire study, the mean percentage of migration inhibition from control chickens was negative; i.e., the migration areas were larger in the presence of P P D than in the absence of P P D ( - 9 . 2 9 to -44.91% migration inhibition). For the 10 wk after immunization, the mean percentage of migration inhibition from immunized birds was -14.23% to 65.00% (Fig. 1). During the first 4 wk postinjection, 11 leukocyte samples from 5 chickens with negative wattle tests had positive L M I T ; however, 27 of 28 tests from 10 birds with positive wattle tests had significant LMIT. Ten chickens not sensitized to myeobacteria were P P D wattle tested at weekly intervals during the first 4 wk of study. In the process of this testing, these chickens apparently became immune to PPD. One of these chickens had 25.5% migration inhibition when tested by L M I T during the second week. During the first 8 wk of the study, 9 of 54 L M I T from these chickens had 20% or more migration inhibition (Table 1). None of these chickens had positive intradermal wattle tests. When tested for the presence of cytophilic antibodies 10 wk after the initial P P D
338
VLAOVIC, BUENING AND LOAN
1oo 90 80 o
70
60 5O 4o
~.
30
./ / WEEKSPOSTINJECTION
Fro. 1. Comparison of leukocyte migration inhibition tests ( 0 O ) with wattle tests (X X ) for delayed hypersensitivity in mycobacterial sensitized chickens.
injection, 7 of 10 sera from these PPD immune chickens had cytophilic antibodies. Only one chicken sensitized to mycobacteria had cytophilic antibody (Table 2). To differentiate positive LMIT due to CMI from those due to cytophilic antibodies, an additional treatment with anti-chicken IgG was introduced (Table 3). Normal chicken peripheral blood leukocytes with passively acquired cytophilic antibody were significantly inhibited in their migration in the presence of PPD. After treatment with rabbit anti-chicken IgG, migration inhibition of peripheral blood leukocytes with cytophilic antibodies was reduced to insignificant levels. Migration inhibition of peripheral blood leukocytes from chickens immunized with Freund's complete adjuvant were not affected. DISCUSSION Delayed hypersensitivity in the chicken as measured by wattle tests (positive test = 100~ increase in thickness) was first detected 2 wk after injection of TABLE 1 INHIBITION OF MIGRATION OF LEUKOCYTES FROM TUBERCULIN IMMUNE CHICKENS a IN THE PRESENCE OF P P D
Week of test
Number with significant LM ITb/number tested
1~ 2c 3c
0/7 1/10 2/10
4c
1/8
6 8
3/10 2/9
a Chickens were not sensitized to mycobacteria. b Significant L M I T = 20% or more migration inhibition. c Wattle injections with 10 ~g P P D in 0.1 ml of Puck's saline.
LEUKOCYTE MIGRATION I N H I B I T I O N I N T H E C H I C K E N
339
TABLE 2 EFFECT OF SERA FROM MYCOBACTERIA SENSITIZED AND PPD IMMUNE CHICKENS ON THE LEUKOCYTE MIGRATION INHIBITION TEST (LMIT)
Serum source
Number of sera with significant LMIT*/number of sera tested
Normal chicken (Autologous)a Normal chicken (Homologous)~ PPD immunechickenb Mycobacteria sensitizedchicken
0/9 0/9 7/10 1/10
a Sera from normal chickens. bSera tested at 10 wk after initial PPD injection. c SignificantLMIT = 20% or more migrationinhibition. Freund's complete adjuvant. Four weeks after injection, 50% of the chickens had developed delayed hypersensitivity. All chickens became hypersensitive by the sixth postinjection week (Fig. 1). Footpad inoculation in the mycobacteria sensitized chickens resulted in an inflammatory response that was initially observed at 1 wk postinjection. This reaction was prominent during the following 3 wk and then subsided. Failure to obtain significant inhibition of migration of peripheral blood leukocytes in 18 chickens during the first 4 wk postinjection could be partially attributed to the accumulation of sensitized leukocytes in the area of inflammation. Similar effects were observed when wattle testing preceded the lymphoid cell harvest by 18-24 hr (4). In studies of CMI, Fauser et al. (6) replaced capillary tubes in the migration inhibition test with drops of cell suspension approximately 4 mm in diameter placed in plastic Petri dishes. Using this technique, valuable data on the role of CMI and humoral antibodies in Marek's disease were obtained. In experiments reported here, some peripheral blood leukocytes (up to 30%) from the tuberculin immune group (not sensitized to mycobacteria) were inhibited in their migration in the presence of PPD (Table 2). Since none of these birds had positive wattle tests at any time during the experiment, the presence of cytophilic antibodies was suspected and later confirmed. Ten weeks after initial wattle testing, 70% of the serum samples from these chickens contained sufficient quantities of eytophilic antibodies to cause normal chicken peripheral blood leukocytes to be inhibited in the presence of PPD (Table 3). Zwilling et al. (3) were unable to detect cytophilic antibody armed cells following the intravenous injection of chickens with bovine gamma globulin. However, in their study four to six sera from chickens with precipitating antibodies to PPD inhibited normal cell migration in the presence of PPD. Immunization schedules that usually lead to the appearance of CMI can produce cytophilic antibodies which are capable of inhibiting the migration of normal macrophages (10). Sera from human patients allergic to penicillin rendered leukocytes from nonallergic individuals susceptible to inhibition when exposed to penicillin antigen (13). The substance involved in this reaction was identified as an antibody. Migration inhibition due to CMI or cytophilic antibodies was indistinguishable by L M I T alone. Treatment with rabbit anti-chicken IgG differentiated these two types of migration inhibition. Rabbit anti-chicken IgG rendered migration inhibition
340
:
VLAOVIC~BUENING AND LOAN TABLE 3
EFFECT OF RABBIT ANTI-CHICKEN IgG ON TKE MIGRATION OF CHICKEN PERIPHERAL BLOOD LEUKOCYTES Reaction mixture
Percentage of migration inhibition
LM IT pos./ no. tested
Normal leukocytes + PPD~ Normal leukocytes
--0.7, --9.5
0/2
Normal leukocytes + cyt. Ab b + PPD Normal leukocytes + cyt. Ab
35.5, 32.5, 34.1
3/3
Normal leukocytes + NChS * + PPD Normal leukocytes + NChS
-21.6, --2.2
0/2
Normal leukocytes + cyt. Ab + Anti IgG d + PPD Normal leukocytes + cyt. Ab + Anti IgG
--6.4, --2.1
0/2
Normal leukocytes + cyt. Ab + NRS ~ + PPD Normal leukocytes + Ab + NRS
29.8, 25.6
2/2
Normal leukocytes + NChS + NRS + PPD Normal leukocytes + NChS + NRS
--2.7, --9.3
0/2
Cyt. Ab armed leukocyteJ + PPD Cyt. Ab armed leukocytes
31.0, 25.2, 24.5
3/3
Cyt. Ab armed leukocytes + Anti IgG + PPD Cyt. Ab armed leukocytes q- Anti IgG
8.0, 3.7, --9.6
0/3
Cyt. Ab armed leukocytes q- NRS -t- PPD Cyt. Ab armed leukocytes + NRS
23.2, 24.7, 23
3/3
Sensitizedg leukocytes + PPD Sensitized leukocytes
21.6, 25.0, 24.7, 23.8, 24.3, 10.0, 8.0
5/7
Sensitized leukocytes + Anti IgG + PPD Sensitized leukocytes + Anti IgG
24.3, 24.4, 24.3, 23.1, 22.7, 10.2, 9.1
5/7
Sensitized leukocytes + NRS + PPD Sensitized leukocytes + NRS
35.9, 32.6, 34.2, 32.7, 29.1, 21.2, 20.4
7/7
PPD = purified protein derivative of tuberculin. b Cyt. Ab = Cytophilie antibody from PPD tested birds with significant leukocyte migration inhibition test (LMIT). c NChS = normal chicken serum. d Anti IgG = rabbit anti-chicken IgG. e NRS = normal rabbit serum. s Cyt. Ab armed leukocytes = leukocytes from PPD immune chickens with significant LMIT. g Sensitized leukocytes = leukocytes from chickens injected with Freund's complete adjuvant. of cytophilic a n t i b o d y - p r e t r e a t e d n o r m a l p e r i p h e r a l blood leukocytes insignificant, and abolished the m i g r a t i o n inhibition of cytophilic antibody a r m e d leukocytes. I t did not affect m i g r a t i o n inhibition due to C M I ( T a b l e 4 ) . T h i s is further evidence that chicken " T " lymphocytes do not c a r r y significant a m o u n t s of surface i m m u n o globulin d e t e r m i n a n t s ( 1 4 ) , and are not affected by rabbit anti-chicken I g G . REFERENCES 1. Morita, C., and Soekawa, N., Poultry Sci., 50, 1503-1505, 1971. 2. Morita, C., and Soekawa, M., Poultry Scl. 51, 1133-1136, 1972.
LEUKOCYTE MIGRATION INHIBITION IN THE CHICKEN 3. 4. 5. 6.
341
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