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Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle
Veterinary Immunology and Immunopathology, 4 (1983) 375--385 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
375
APPLICATION OF INDOMETHACIN AS A POTENTIATOR OF LYMPHOCYTE BLASTOGENESIS IN BRUCELLA ABORTUS EXPOSED CATTLE
J.M.B. KANEENEI , R.K. ANDERSON2, D.W. JOHNSON~ and C.C. MUSCOPLAT3 iDepartment of Large Animal C l i n i c a l Sciences, Michigan State University, East Lansing, MI 48824, U.S.A. 2Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, MN 55455, U.S.A. 3Department of Large Animal C l i n i c a l Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, U.S.A. (Accepted 21 July 1982) ABSTRACT Kaneene, J.M.B., Anderson, R.K., Johnson, D.W. and Muscoplat, C.C., 1983. Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed c a t t l e . Vet. Immunol. Immunopathol., 4: 375-385. Lymphocytes from Brucella abortus f i e l d strain infected, strain 19 vaccinated, non-exposed and f i e l d strain infected, but immunologically unresponsive c a t t l e were incubated with B. abortus antigen and indomethacin. There were s i g n i f i c a n t increases (P < 0.005) in the blastogenic responses, as measured by [3H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to B. abortus antigen were potentiated by indomethacin in both B. abortus exposed and nonexposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was s i g n i f i c a n t l y greater (P < 0.005) than that in non-exposed lymphocytes. A d d i t i o n a l l y , indomethacin s i g n i f i c a n t l y potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle. INTRODUCTION Indomethacin (INDO) has been shown by these laboratories to potentiated mitogen- and antigen-induced in v i t r o lymphocyte stimulation responses (LSR) in M~cobacterium bovis sensitized c a t t l e (Muscoplat, et a l . , 1978).
The poten-
t i a t i o n of the antigen-induced LSR by INDO was reported to be immunologically s p e c i f i c and t h i s observation may be applied to study many diagnostic and applied immunological problems. In t h i s study, we describe the application of the antigen-induced LSR potentiation by INDO in two specific diagnostic
0165-2427/83/$03.00
© 1983 Elsevier Science Publishers B.V.
376
problems in bovine b r u c e l l o s i s ; unresponsiveness and anergy. In a series of papers, we reported on the usefulness of the in v i t r o lymphocyte stimulation test (LST) in the diagnosis of bovine (Kaneene, et a l . , 1978a, 1978b, 1978c, 1978d, 1978e, 1978f) and swine (Kaneene, et a l . , 1978g) br uc e llo sis.
Using LST r e s u l t s , we were able to d i f f e r e n t i a t e B. abortus f i e l d strain
infected c a t t l e from those that were vaccinated with B. abortus s t r a i n 19 vaccine (Kaneene, et a l . , 1978a, 1978b, 1978c).
A d d i t i o n a l l y , the LST detected B.
abortus f i e l d strain infected c a t t l e early in the incubation period before any serological test became p o s i t i v e (Kaneene, et a l . , 1978f).
Using the LST
r e s u l t s , we could not, however, d i f f e r e n t i a t e c a t t l e that were vaccinated with B. abortus s t r a i n 19 (U.S. Department of A g r i c ult u r e o f f i c i a l those that were not exposed to brucellae. detect anergic animals. determine:
vaccine) from
Secondly using the LST, we could not
The specific objectives of this study were to
I) whether INDO produces d i f f e r e n t levels of p o t e n t i a t i o n of
Brucella-induced LSR in B. abortus f i e l d s t r a i n , strain 19 vaccinated and nonexposed c a t t l e lymphocytes; 2) whether INDO potentiates Brucella-induced LSR in lymphocytes from B. abortus infected but non-responsive c a t t l e (anergic cattle). MATERIAL AND METHODS Animals:
Animals were grouped, according to exposure experiences to B.
abortus, into 4 groups. cows.
Group I (Table I) was composed of 16 adult milking
Each of these animals was n a t u r a l l y infected with f i e l d - s t r a i n of B.
abortus biotype 1 (as determined by i s o l a t i o n from t h e i r milk).
Group 2 (Table
I I ) consisted of 8 milking cows that had been vaccinated (at the age of 4 months) with B. abortus s t r a i n 19. non-exposed c a t t l e ( c o n t r o l s ) .
Group 3 (Table I I ) consisted of 8 Brucella
Group 4 (Table I I I )
(anergic animals) described as follows:
consisted of 4 milking cows
Cow 69257 was n a t u r a l l y infected with
B. abortus biotype 1 and isolates were repeatedly made from her milk, cow number 3416 was vaccinated with B. abortus s t r a i n 19.
Both of these animals
(cows 69257 and 3416) were giving p o s i t i v e results on both serological tests and LST f o r over one year.
Three months p r i o r to this study, these animals
went negative on serological and lymphocyte stimulation tests even though bruc e l l a e were repeatedly i s o l a t e d from t h e i r milk during that time. mals were termed anergic c a t t l e .
These ani-
Animals F03 and F012 were milking cows from
the f i e l d and B. abortus biotype 1 was isolated from them. Preparation of lymphocyte cultures:
Blood was obtained via jugular veni-
puncture and placed into heparinized tubes.
Mononuclear c e l l s were separated
by c e n t r i f u g a t i o n over Ficoll-Hypaque and subsequently washed twice with Hanks' balanced salt solution (HBSS). Mononuclear c e l l s were counted and suspended to a concentration of 1.5 x 106 c e l l s per ml in RPMI-1640 medium containing 25 mM
377
HEPES buffer, 15% f e t a l bovine serum, and gentamicin (50 ~g/ml).
Cells were
cultured in flat-bottom m i c r o t i t e r plates, 3 x 105 c e l l s in 200 ul. Concanavalin A (ConA) (Miles Yeda, Rehovot, I s r a e l ) was used, at a concentration of 2.0 ~g/well, as the mitogen and B. abortus soluble antigen (BASA), at a concentration of 4.4 ~g/wel], was used as the specific antigen. The preparation and preliminary chemical analysis of this antigen has been reported (Kaneene, et a l . , 1978a).
Indomethacin (Sigma Co., St. Louis, MO),
was dissolved in 4% NaHCO3 then diluted in HBSS and was added to appropriate cultures at a concentration of 1.0 ~g/well.
Cultures were incubated at 37 ° C
in a 5% CO2 humidified a i r atmosphere for a total of 5 days.
Cultures were
pulsed with [3H] thymidine ( i . 0 ~Ci, 6.0 Ci/mmole; Research Products I n t e r n a t i o n a l , Elk Grove V i l l a g e , IL), 16 to 18 hours prior to harvesting. Cultures were terminated by harvesting c e l l s onto glass f i b e r f i l t e r s
(Skatron
Cell Harvester; Flow Laboratories, Rockville, MD) and were counted for 3H a c t i v i t y in a l i q u i d s c i n t i l l a t i o n spectrometer.
Cultures were performed in
triplicate. Expresssion of results - results were expressed in three ways:
1) Stimulation Index (SI)
= mean counts per minute (CPM) of t r i p l i c a t e cultures with antigen (AG) or mitogen, mean CPM of t r i p l i c a t e culture without AG or mitogen.
2) Change in CPM (~ CPM)
=Fmean CPM of t r i p l i c a t e ] Lcultures with AG
3)
= mean CPM in cultures with
Net Potentiation CPM
_
FmeanCPM of t r i p l i c a t e - ] Lcultures without AG _]
IAG + INDO)-INDO 1
iAG - CONTROL~
S t a t i s t i c a l analysis of the data was conducted using a student's t - t e s t . RESULTS The complete raw data (CPM) from 3 groups of animals are presented (Tables I , I I and I I I ) .
Using Sl, i t can be seen (Fig. I ) that BASA-induced LSR were
s i g n i f i c a n t l y higher (P < 0.005) in lymphocytes from group i c a t t l e than those in lymphocytes from either groups 2 or 3 c a t t l e . The BASA-induced LSR in lymphocytes from groups 2 and 3 c a t t l e were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05).
Figure 2 shows the comparison of LSR induced by BASA alone and those
induced by BASA plus INDO.
In each groups of animals (Fig. 2), the
BASA+INDO-induced LSR were higher than those induced by LSR only.
Indomethacin
induced s i g n i f i c a n t net potentiation of BASA-induced LSR in B. abortus exposed
378
.B. a,,bortus Field Strain Infected B. abortus St. 19 Vaccinated L---] Non-Exposed )< Q; X~ c-
Q
Number Tested
c-
.(2_ E
Groups
Figure 1 Lymphocyte b lastogenic responses induced by B. abortus antigen and indomethacin in B. abortus sensitized and non-exposed cattle lymphocytes. Vertical small bars [I] represent standard error of the means.
379
TABLE I Potentiation of ConA and Brucella abortus Blastogenic Responses in Brucella abortus Infected Cattle Lymphocytes Treated with Indomethacin POTENTIATION (CPM)a Animals (Group 1)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CON A
CON A + INDO
8ASAb
BASA + INDO
INDO
BASA CONTROL CONT= SI
19,789 82,851 43,207 48,089 4,829 7,020 20,137 21,815 31,731 ND 27,715 ND 44,276 20,234 15,730 47,151
28,977 96,465 51,614 59,980 14,525 9,587 25,047 13,234 38,810 ND 29,785 ND 36,963 36,001 18,477 49,675
3,188 44,399 8,065 7,274 616 1,510 1,645 9,621 4,661 1,978 480 511 901 841 760 780
19,133 93,459 23,079 18,880 2,166 9,944 4,840 13,681 16,701 6,119 3,714 1,018 5,544 2,101 8,202 34,415
431 783 2 , 8 7 2 8,886 314 659 399 126 285 113 1,678 196 174 178 170 166 174 117 ND 135 102 122 172 150 290 137 392 194 2,097 194 1,301 129
4.1 5.0 12.2 57.7 5.5 7.7 9.2 58.0 39.8 14.7 3.9 3.4 6.6 4.3 3.9 6.1
aCounts per minute of t r i p l i c a t e cultures. bBrucella abortus soluble antigen. cattle lymphocytes and only minimal net potentiation in lymphocytes from nonexposed cattle (Fig. 3).
Additionally, INDO potentiated significantly
BASA-induced LSR in lymphocytes from 4 anergic cows (Table I l l ) .
Similarly,
INDO potentiated ConA-induced blastogenesis (Tables I - I l l ) . DISCUSSION We have demonstrated in this study that INDO significantly potentiates the BASA-induced LSR in Brucella exposed but non-responsive cattle lymphocytes. The mechanisms by which INDO potentiates antigen-induced LSR is said to be an indirect one. Many investigators have shown that prostaglandins suppress mitogen-induced (Goodwin, et a l . , 1977a, 1977b, 1978; Mihas, et a l , , 1975) and antigen-induced immuno responses (Ferraris, e t a ] . , 1974; Webb, et a l . , 1977; Zimecki, et a l . , 1976). Webb and Nowowiejski (1978), and Goodman, et al. (1978), have reported that prostaglandins induce short-lived suppressor cells
380
18 16
14 12
Brucella Antigen
J~
Brucella Antigen, Indomethacin Number Tested
/ / /
I
0 x E
7 / / /
D
lO
/ / /
8
// / /
6
// / /
n U
2
o
Infected
-
-
Vacci hated
Non- Exposed
Figure 2 Net potentiation of B. abortus antigen-induced lymphocyte blastogenic responses by indomethacin in B. abortus sensitized and non-exposed cattle lymphocytes. Vertical small bars [I] represent standard error of the mean~
381
TABLE I I P o t e n t i a t i o n of ConA and Brucella abortus Blastogenic Responses in Brucella abortus Strain 19 Vaccine Exposed and Non-Exposed Cattle Lymphocytes Treated with Indomethacin
POTENTIATION (CPM)a
Animals
CON A
CON A + INDO
BASAb
BASA + INDO
INDO
BASA CONTROL CONT= SI
B. abortus vaccinates c (Group 2) 17 33 695 937 945 947 952 959
379 296 306 283 377 371 197 356
418 826 616 288 110 467 331 363
437,946 404,107 368,764 416,576 433,008 419,515 368,196 442,063
1,410 4,516 4,683 1,659 14,097 2,044 3,544 822
3,485 13,890 24,178 11,976 24,393 4,812 12,112 2,144
3,016 14,161 15,855 3,328 20,018 1,845 9,419 1,784
1,491 4,333 5,581 853 13,683 1,538 4,264 690
1.0 1.0 0.8 1.9 1.0 1.3 0.8 1.2
833 191 181 287 154 703 831 992
695 524 1,149 430 344 1,409 1,973 3,537
352 290 1,097 1,203 207 1,200 1,748 2,028
271 137 184 130 93 776 660 568
3.1 1.4 1.0 2.2 1.7 0.9 1.3 1.8
Non-exposed controls (Group 3) 5 18 19 24 25 179 688 800
13,664 31,308 130,704 54,590 20,644 119,102 355,331 290,082
140,142 19,493 161,958 44,751 82,427 342,010 448,603 333,402
acounts per minute of t r i p l i c a t e cultures. bBrucella abortus soluble antigen. CVaccinated with B. abortus s t r a i n 19 vaccine. which in turn release a suppressor product which w i l l lead to immune suppression.
Since indomethacin is a potent prostaglandin synthesis i n h i b i t o r ,
and since prostaglandins have been shown to suppress immunological responses, it
is reasonable to assume that in the present study indomethacin produced
immunological enhancement by i n h i b i t i n g endogenous production of prostaglandins by mononuclear c e l l s in cultures,
This allowed the antigen-induced specific
blastogenesis responses to go on without suppression.
The net r e s u l t was a
s p e c i f i c p o t e n t i a t i o n of antigen-induced lymphocyte s t i m u l a t i o n responses. Using our routine (Sl) analysis of the data, we could not d i f f e r e n t i a t e B.
382
12
10
B. a b o r t u s Field Strain Infected B. a b o r t u s Strain 19 Vaccinated
o
E n L) -r
r-~
Non-Exposed
Q
N u m b e r Tested
._o .o_ ¢-
no z
iiiiiiiiiiiiiiiiiiiii iii!iiiiiiiiiiiiiiiiiiii;! !i!ii!iiiiii!i!iiiiiii!i!iiii!iiil iiiiiii~i~iiiiiiiiiiiiii!ii~i~iiii
Groups
Figure 3 N e t p o t e n t i a t i o n of B. abortus a n t i g e n - i n d u c e d lymphocyte b l a s t o g e n i c r e s p o n s e s by i n d o m e t h a c i n in B. abortus s e n s i t i z e d and n o n - e x p o s e d cattle lymphocyteso V e r t i c a l small bars (I) represent standard error of the mean~
383
TABLE I l l Potentiation of ConA and Brucella abortus-lnduced Blastogenic Responses by Indomethacin in Lymphocytes from B. abortus ExposedBut NonresponsiveCattle
POTENTIATION (CPM)a
Date
7/27/78 8/24/78 9/1/78
Animal No.
3416
CON A
CON A + INDO BASA
BASA + INDO
BASA INDO CONTROL CONT= SI
11,702 ND 887 ND ND 212,719 310,281 1,550 10,518 1,714 47,151 49,675 780 3 , 4 4 9 1,301
612 716 329
1.5 2.2 2.4
69257 7/27/78 8/24/78 9/I/78 9/1/78 9/1/78
F03 F012
10,903 ND 28,989 366,071 36,963 44,276
774 607 901
ND 4,939 5,544
ND 847 446
534 428 381
1.5 1.4 2.4
18,894 177,714 37,975 45,635
910 293
5,847 2,391
284 467
525 165
1.7 1.8
INDO = Indomethacin. CON A = Concanavalin A. BASA = Brucella abortus soluble antigen. aCounts per minute of t r i p l i c a t e cultures. abortus strain 19 vaccinated cattle from those that were not exposed (Fig. 1). Similarly, we could not detect animals numbers 3416 and 69257 {anergic) prior to the application of INDO (Table I I ) .
WhenBASA + INDO model was applied, we
were able to d i f f e r e n t i a t e vaccinated from non-exposedcattle and we were able to detect LSR in anergic cattle.
Animal number 5 in group 3 (non-exposed) had
a false positive response (SI ~ 3.0) when BASAalone was used. However, after application of INDO, the SI was below 3.0. Vaccination of cattle with B. abortus strain 19 (U.S.D.A. o f f i c i a l vaccine), is l i k e l y to continue for a long time in the United States.
This vaccine,
however, causes some diagnostic problems in that some animals retain persistent serum antibody t i t e r s which makes i t d i f f i c u l t to serologically d i f f e r e n t i a t e them from cattle that have infections with f i e l d strains of Brucella.
On the
other hand, most vaccinated animals we have tested in our laboratory have given minimal or no LSR. These two problems are further complicated by the fact that some animals lose their i d e n t i f i c a t i o n during their movement to other farms or markets. Without i d e n t i f i c a t i o n and proper history of the animals, epidemiologists in the f i e l d are not sure of what type of exposure such animals have had.
384
Since the vaccine used gives significant protection i t is l i k e l y to stay, what is needed is a test (or tests) that would differentiate cattle according to the type of Brucella exposure experiences ( i . e . , whether infected with f i e l d strains, vaccinated or non-exposed). The data presented in this study suggests that the in v i t r o LST using antigen+INDO model may have significant diagnostic application in bovine brucellosis.
Further studies should be conducted where a
large number of Brucella exposed cattle w i l l be used to strengthen our findings. ACKNOWLEDGEMENTS Supported in part by the U.S. Department of A g r i c u l t u r e , Veterinary Services and Science Education Administration, and the Minnesota A g r i c u l t u r a l Experiment Station. We thank Diane Klausner for her technical help.
REFERENCES Ferraris, V.A. and Derubertis, F.R., 1974. Release of prostaglandin by mitogen-induced-stimulated leukocytes in culture. J. Clin. Invest. 54 : 378-386. Goodwin, J.S., Bankhurst, A.D. and Messner, R.P., 1977a. Suppression of human T-cell mitogenesis by prostaglandin: evidence of a prostaglandin-producing suppressor c e l l . J. Exp. Med. 146 : 1719-1734. Goodwin, J.S., Messner, R.P., Burkhurst, A.D., Peake, G.T., Saiki, J.H. and Williams, J r . , R.C., 1977b. N. Engl. J. Med. 297 : 963-968. Goodwin, J.S., Messner, R.P. and Peake, G.T., 1978. Prostaglandin suppression of mitogen-stimulated lymphocytes in v i t r o . Changes with mitogen dose and preincubation. J. Clin. Invest. 62 : 753-760. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K., Angus, R.D., Pietz, D.E. and Muscoplat, C.C., 1978a. Kinetics of in v i t r o bovine lymphocyte immunostimulation with a Brucella abortus antigen. Am. J. Vet. Res. 39(2) : 235-239. Kaneene, J.M.B., ~ D.W., Anderson, R.K., Angus, R.D., Pietz, D.E. and Muscoplat, C.C., 1978b. Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19. Am. J. Vet. Res. 39(4) : 585-589. Kaneene, J.M.B., Anderson, R.K., Johnson, D.W., Muscoplat, C.C., Nicoletti, P., Angus, R.D., Pietz, D.E. and Klausner, D.J., 1978c. Whole-blood lymphocyte stimulation assay for measurement of cell-mediated immune responses in bovine brucellosis. J. Clin. Microbiol. 7 : 550-557. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K. and Muscoplat, C.C., 1978d. Comparison of s e n s i t i v i t y and s p e c i f i c i t y of purified lymphocytes and wholeblood in v i t r o lymphocyte stimulation assays in detection of Brucella a b o r t u - s - i ~ i o n in cattle. J. Clin. Microbiol. 8 : 396-40T. Kaneene, J.M.B., Anderson, R.K., Johnson, D.W. and Muscoplat, C.C., 1978e. Brucella antigen preparations for in v i t r o lymphocyte immunostimulation assays in bovine brucellosis. Infect. and Immun. 22(2) : 486-491. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K. and Muscoplat, C.C., 1978f. U t i l i z a t i o n of a specific in v i t r o immunostimulation assay as an aid in detection of B r u c e l l a - i n f e ~ e ~ t l e not detected by serological tests. J. Clin. Microbiol. 8 : 512-515.
385
Kaneene, J.M.B., Anderson, R.K., Johnson, D.W., Angus, R.D., Muscoplat, C.C., Pietz, D.E., Vanderwagon, L.C. and Sloane, E.E., 1978g. Responses in swine from a herd infected with Brucella suis. Am. J. Vet. Res. 39(10) : 1607-1611. Mihas, A.A., Gibson, R.G. and Hirschowitz, B . I . , 1975. Suppression of lymphocyte transformation by lb. (16) diamethyl prostaglandin E2 and unsaturated f a t t y acids. Proc. Soc. Exp. Biol. Med. 149 : 1026-1028. Muscoplat, C.C., Rakich, P.M., Thoen, C.O. and Johnson, D.W., 1978. Enhancement of lymphocyte blastogenic and delayed h y p e r s e n s i t i v i t y skin responses by indomethacin. Infect. and Immun. 20 : 627-629. Webb, D.R., Nowowiejski, I . , Dauphinne, M. and Talal, N., 1977. Antigeninduced alterations in splenic prostaglandin and cyclic nucleotiole levels in N2B mice. J. Immunol. 118 : 446-450. Webb, D.R., Rogers, T.I. and Nowowiejski, I . , 1979. Endogeneous prostaglandin synthesis and the control of lymphocyte function. Ann. NY. Acad. Sci. 332 : 262-270. Zimecki, M. and Webb, D.R°, 1976. The regulation of the immune response to Tindependent antigens by prostaglandins and B-cells. J. Immunol. 117 : 2158-2164.
375
APPLICATION OF INDOMETHACIN AS A POTENTIATOR OF LYMPHOCYTE BLASTOGENESIS IN BRUCELLA ABORTUS EXPOSED CATTLE
J.M.B. KANEENEI , R.K. ANDERSON2, D.W. JOHNSON~ and C.C. MUSCOPLAT3 iDepartment of Large Animal C l i n i c a l Sciences, Michigan State University, East Lansing, MI 48824, U.S.A. 2Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, MN 55455, U.S.A. 3Department of Large Animal C l i n i c a l Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, U.S.A. (Accepted 21 July 1982) ABSTRACT Kaneene, J.M.B., Anderson, R.K., Johnson, D.W. and Muscoplat, C.C., 1983. Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed c a t t l e . Vet. Immunol. Immunopathol., 4: 375-385. Lymphocytes from Brucella abortus f i e l d strain infected, strain 19 vaccinated, non-exposed and f i e l d strain infected, but immunologically unresponsive c a t t l e were incubated with B. abortus antigen and indomethacin. There were s i g n i f i c a n t increases (P < 0.005) in the blastogenic responses, as measured by [3H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to B. abortus antigen were potentiated by indomethacin in both B. abortus exposed and nonexposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was s i g n i f i c a n t l y greater (P < 0.005) than that in non-exposed lymphocytes. A d d i t i o n a l l y , indomethacin s i g n i f i c a n t l y potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle. INTRODUCTION Indomethacin (INDO) has been shown by these laboratories to potentiated mitogen- and antigen-induced in v i t r o lymphocyte stimulation responses (LSR) in M~cobacterium bovis sensitized c a t t l e (Muscoplat, et a l . , 1978).
The poten-
t i a t i o n of the antigen-induced LSR by INDO was reported to be immunologically s p e c i f i c and t h i s observation may be applied to study many diagnostic and applied immunological problems. In t h i s study, we describe the application of the antigen-induced LSR potentiation by INDO in two specific diagnostic
0165-2427/83/$03.00
© 1983 Elsevier Science Publishers B.V.
376
problems in bovine b r u c e l l o s i s ; unresponsiveness and anergy. In a series of papers, we reported on the usefulness of the in v i t r o lymphocyte stimulation test (LST) in the diagnosis of bovine (Kaneene, et a l . , 1978a, 1978b, 1978c, 1978d, 1978e, 1978f) and swine (Kaneene, et a l . , 1978g) br uc e llo sis.
Using LST r e s u l t s , we were able to d i f f e r e n t i a t e B. abortus f i e l d strain
infected c a t t l e from those that were vaccinated with B. abortus s t r a i n 19 vaccine (Kaneene, et a l . , 1978a, 1978b, 1978c).
A d d i t i o n a l l y , the LST detected B.
abortus f i e l d strain infected c a t t l e early in the incubation period before any serological test became p o s i t i v e (Kaneene, et a l . , 1978f).
Using the LST
r e s u l t s , we could not, however, d i f f e r e n t i a t e c a t t l e that were vaccinated with B. abortus s t r a i n 19 (U.S. Department of A g r i c ult u r e o f f i c i a l those that were not exposed to brucellae. detect anergic animals. determine:
vaccine) from
Secondly using the LST, we could not
The specific objectives of this study were to
I) whether INDO produces d i f f e r e n t levels of p o t e n t i a t i o n of
Brucella-induced LSR in B. abortus f i e l d s t r a i n , strain 19 vaccinated and nonexposed c a t t l e lymphocytes; 2) whether INDO potentiates Brucella-induced LSR in lymphocytes from B. abortus infected but non-responsive c a t t l e (anergic cattle). MATERIAL AND METHODS Animals:
Animals were grouped, according to exposure experiences to B.
abortus, into 4 groups. cows.
Group I (Table I) was composed of 16 adult milking
Each of these animals was n a t u r a l l y infected with f i e l d - s t r a i n of B.
abortus biotype 1 (as determined by i s o l a t i o n from t h e i r milk).
Group 2 (Table
I I ) consisted of 8 milking cows that had been vaccinated (at the age of 4 months) with B. abortus s t r a i n 19. non-exposed c a t t l e ( c o n t r o l s ) .
Group 3 (Table I I ) consisted of 8 Brucella
Group 4 (Table I I I )
(anergic animals) described as follows:
consisted of 4 milking cows
Cow 69257 was n a t u r a l l y infected with
B. abortus biotype 1 and isolates were repeatedly made from her milk, cow number 3416 was vaccinated with B. abortus s t r a i n 19.
Both of these animals
(cows 69257 and 3416) were giving p o s i t i v e results on both serological tests and LST f o r over one year.
Three months p r i o r to this study, these animals
went negative on serological and lymphocyte stimulation tests even though bruc e l l a e were repeatedly i s o l a t e d from t h e i r milk during that time. mals were termed anergic c a t t l e .
These ani-
Animals F03 and F012 were milking cows from
the f i e l d and B. abortus biotype 1 was isolated from them. Preparation of lymphocyte cultures:
Blood was obtained via jugular veni-
puncture and placed into heparinized tubes.
Mononuclear c e l l s were separated
by c e n t r i f u g a t i o n over Ficoll-Hypaque and subsequently washed twice with Hanks' balanced salt solution (HBSS). Mononuclear c e l l s were counted and suspended to a concentration of 1.5 x 106 c e l l s per ml in RPMI-1640 medium containing 25 mM
377
HEPES buffer, 15% f e t a l bovine serum, and gentamicin (50 ~g/ml).
Cells were
cultured in flat-bottom m i c r o t i t e r plates, 3 x 105 c e l l s in 200 ul. Concanavalin A (ConA) (Miles Yeda, Rehovot, I s r a e l ) was used, at a concentration of 2.0 ~g/well, as the mitogen and B. abortus soluble antigen (BASA), at a concentration of 4.4 ~g/wel], was used as the specific antigen. The preparation and preliminary chemical analysis of this antigen has been reported (Kaneene, et a l . , 1978a).
Indomethacin (Sigma Co., St. Louis, MO),
was dissolved in 4% NaHCO3 then diluted in HBSS and was added to appropriate cultures at a concentration of 1.0 ~g/well.
Cultures were incubated at 37 ° C
in a 5% CO2 humidified a i r atmosphere for a total of 5 days.
Cultures were
pulsed with [3H] thymidine ( i . 0 ~Ci, 6.0 Ci/mmole; Research Products I n t e r n a t i o n a l , Elk Grove V i l l a g e , IL), 16 to 18 hours prior to harvesting. Cultures were terminated by harvesting c e l l s onto glass f i b e r f i l t e r s
(Skatron
Cell Harvester; Flow Laboratories, Rockville, MD) and were counted for 3H a c t i v i t y in a l i q u i d s c i n t i l l a t i o n spectrometer.
Cultures were performed in
triplicate. Expresssion of results - results were expressed in three ways:
1) Stimulation Index (SI)
= mean counts per minute (CPM) of t r i p l i c a t e cultures with antigen (AG) or mitogen, mean CPM of t r i p l i c a t e culture without AG or mitogen.
2) Change in CPM (~ CPM)
=Fmean CPM of t r i p l i c a t e ] Lcultures with AG
3)
= mean CPM in cultures with
Net Potentiation CPM
_
FmeanCPM of t r i p l i c a t e - ] Lcultures without AG _]
IAG + INDO)-INDO 1
iAG - CONTROL~
S t a t i s t i c a l analysis of the data was conducted using a student's t - t e s t . RESULTS The complete raw data (CPM) from 3 groups of animals are presented (Tables I , I I and I I I ) .
Using Sl, i t can be seen (Fig. I ) that BASA-induced LSR were
s i g n i f i c a n t l y higher (P < 0.005) in lymphocytes from group i c a t t l e than those in lymphocytes from either groups 2 or 3 c a t t l e . The BASA-induced LSR in lymphocytes from groups 2 and 3 c a t t l e were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05).
Figure 2 shows the comparison of LSR induced by BASA alone and those
induced by BASA plus INDO.
In each groups of animals (Fig. 2), the
BASA+INDO-induced LSR were higher than those induced by LSR only.
Indomethacin
induced s i g n i f i c a n t net potentiation of BASA-induced LSR in B. abortus exposed
378
.B. a,,bortus Field Strain Infected B. abortus St. 19 Vaccinated L---] Non-Exposed )< Q; X~ c-
Q
Number Tested
c-
.(2_ E
Groups
Figure 1 Lymphocyte b lastogenic responses induced by B. abortus antigen and indomethacin in B. abortus sensitized and non-exposed cattle lymphocytes. Vertical small bars [I] represent standard error of the means.
379
TABLE I Potentiation of ConA and Brucella abortus Blastogenic Responses in Brucella abortus Infected Cattle Lymphocytes Treated with Indomethacin POTENTIATION (CPM)a Animals (Group 1)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CON A
CON A + INDO
8ASAb
BASA + INDO
INDO
BASA CONTROL CONT= SI
19,789 82,851 43,207 48,089 4,829 7,020 20,137 21,815 31,731 ND 27,715 ND 44,276 20,234 15,730 47,151
28,977 96,465 51,614 59,980 14,525 9,587 25,047 13,234 38,810 ND 29,785 ND 36,963 36,001 18,477 49,675
3,188 44,399 8,065 7,274 616 1,510 1,645 9,621 4,661 1,978 480 511 901 841 760 780
19,133 93,459 23,079 18,880 2,166 9,944 4,840 13,681 16,701 6,119 3,714 1,018 5,544 2,101 8,202 34,415
431 783 2 , 8 7 2 8,886 314 659 399 126 285 113 1,678 196 174 178 170 166 174 117 ND 135 102 122 172 150 290 137 392 194 2,097 194 1,301 129
4.1 5.0 12.2 57.7 5.5 7.7 9.2 58.0 39.8 14.7 3.9 3.4 6.6 4.3 3.9 6.1
aCounts per minute of t r i p l i c a t e cultures. bBrucella abortus soluble antigen. cattle lymphocytes and only minimal net potentiation in lymphocytes from nonexposed cattle (Fig. 3).
Additionally, INDO potentiated significantly
BASA-induced LSR in lymphocytes from 4 anergic cows (Table I l l ) .
Similarly,
INDO potentiated ConA-induced blastogenesis (Tables I - I l l ) . DISCUSSION We have demonstrated in this study that INDO significantly potentiates the BASA-induced LSR in Brucella exposed but non-responsive cattle lymphocytes. The mechanisms by which INDO potentiates antigen-induced LSR is said to be an indirect one. Many investigators have shown that prostaglandins suppress mitogen-induced (Goodwin, et a l . , 1977a, 1977b, 1978; Mihas, et a l , , 1975) and antigen-induced immuno responses (Ferraris, e t a ] . , 1974; Webb, et a l . , 1977; Zimecki, et a l . , 1976). Webb and Nowowiejski (1978), and Goodman, et al. (1978), have reported that prostaglandins induce short-lived suppressor cells
380
18 16
14 12
Brucella Antigen
J~
Brucella Antigen, Indomethacin Number Tested
/ / /
I
0 x E
7 / / /
D
lO
/ / /
8
// / /
6
// / /
n U
2
o
Infected
-
-
Vacci hated
Non- Exposed
Figure 2 Net potentiation of B. abortus antigen-induced lymphocyte blastogenic responses by indomethacin in B. abortus sensitized and non-exposed cattle lymphocytes. Vertical small bars [I] represent standard error of the mean~
381
TABLE I I P o t e n t i a t i o n of ConA and Brucella abortus Blastogenic Responses in Brucella abortus Strain 19 Vaccine Exposed and Non-Exposed Cattle Lymphocytes Treated with Indomethacin
POTENTIATION (CPM)a
Animals
CON A
CON A + INDO
BASAb
BASA + INDO
INDO
BASA CONTROL CONT= SI
B. abortus vaccinates c (Group 2) 17 33 695 937 945 947 952 959
379 296 306 283 377 371 197 356
418 826 616 288 110 467 331 363
437,946 404,107 368,764 416,576 433,008 419,515 368,196 442,063
1,410 4,516 4,683 1,659 14,097 2,044 3,544 822
3,485 13,890 24,178 11,976 24,393 4,812 12,112 2,144
3,016 14,161 15,855 3,328 20,018 1,845 9,419 1,784
1,491 4,333 5,581 853 13,683 1,538 4,264 690
1.0 1.0 0.8 1.9 1.0 1.3 0.8 1.2
833 191 181 287 154 703 831 992
695 524 1,149 430 344 1,409 1,973 3,537
352 290 1,097 1,203 207 1,200 1,748 2,028
271 137 184 130 93 776 660 568
3.1 1.4 1.0 2.2 1.7 0.9 1.3 1.8
Non-exposed controls (Group 3) 5 18 19 24 25 179 688 800
13,664 31,308 130,704 54,590 20,644 119,102 355,331 290,082
140,142 19,493 161,958 44,751 82,427 342,010 448,603 333,402
acounts per minute of t r i p l i c a t e cultures. bBrucella abortus soluble antigen. CVaccinated with B. abortus s t r a i n 19 vaccine. which in turn release a suppressor product which w i l l lead to immune suppression.
Since indomethacin is a potent prostaglandin synthesis i n h i b i t o r ,
and since prostaglandins have been shown to suppress immunological responses, it
is reasonable to assume that in the present study indomethacin produced
immunological enhancement by i n h i b i t i n g endogenous production of prostaglandins by mononuclear c e l l s in cultures,
This allowed the antigen-induced specific
blastogenesis responses to go on without suppression.
The net r e s u l t was a
s p e c i f i c p o t e n t i a t i o n of antigen-induced lymphocyte s t i m u l a t i o n responses. Using our routine (Sl) analysis of the data, we could not d i f f e r e n t i a t e B.
382
12
10
B. a b o r t u s Field Strain Infected B. a b o r t u s Strain 19 Vaccinated
o
E n L) -r
r-~
Non-Exposed
Q
N u m b e r Tested
._o .o_ ¢-
no z
iiiiiiiiiiiiiiiiiiiii iii!iiiiiiiiiiiiiiiiiiii;! !i!ii!iiiiii!i!iiiiiii!i!iiii!iiil iiiiiii~i~iiiiiiiiiiiiii!ii~i~iiii
Groups
Figure 3 N e t p o t e n t i a t i o n of B. abortus a n t i g e n - i n d u c e d lymphocyte b l a s t o g e n i c r e s p o n s e s by i n d o m e t h a c i n in B. abortus s e n s i t i z e d and n o n - e x p o s e d cattle lymphocyteso V e r t i c a l small bars (I) represent standard error of the mean~
383
TABLE I l l Potentiation of ConA and Brucella abortus-lnduced Blastogenic Responses by Indomethacin in Lymphocytes from B. abortus ExposedBut NonresponsiveCattle
POTENTIATION (CPM)a
Date
7/27/78 8/24/78 9/1/78
Animal No.
3416
CON A
CON A + INDO BASA
BASA + INDO
BASA INDO CONTROL CONT= SI
11,702 ND 887 ND ND 212,719 310,281 1,550 10,518 1,714 47,151 49,675 780 3 , 4 4 9 1,301
612 716 329
1.5 2.2 2.4
69257 7/27/78 8/24/78 9/I/78 9/1/78 9/1/78
F03 F012
10,903 ND 28,989 366,071 36,963 44,276
774 607 901
ND 4,939 5,544
ND 847 446
534 428 381
1.5 1.4 2.4
18,894 177,714 37,975 45,635
910 293
5,847 2,391
284 467
525 165
1.7 1.8
INDO = Indomethacin. CON A = Concanavalin A. BASA = Brucella abortus soluble antigen. aCounts per minute of t r i p l i c a t e cultures. abortus strain 19 vaccinated cattle from those that were not exposed (Fig. 1). Similarly, we could not detect animals numbers 3416 and 69257 {anergic) prior to the application of INDO (Table I I ) .
WhenBASA + INDO model was applied, we
were able to d i f f e r e n t i a t e vaccinated from non-exposedcattle and we were able to detect LSR in anergic cattle.
Animal number 5 in group 3 (non-exposed) had
a false positive response (SI ~ 3.0) when BASAalone was used. However, after application of INDO, the SI was below 3.0. Vaccination of cattle with B. abortus strain 19 (U.S.D.A. o f f i c i a l vaccine), is l i k e l y to continue for a long time in the United States.
This vaccine,
however, causes some diagnostic problems in that some animals retain persistent serum antibody t i t e r s which makes i t d i f f i c u l t to serologically d i f f e r e n t i a t e them from cattle that have infections with f i e l d strains of Brucella.
On the
other hand, most vaccinated animals we have tested in our laboratory have given minimal or no LSR. These two problems are further complicated by the fact that some animals lose their i d e n t i f i c a t i o n during their movement to other farms or markets. Without i d e n t i f i c a t i o n and proper history of the animals, epidemiologists in the f i e l d are not sure of what type of exposure such animals have had.
384
Since the vaccine used gives significant protection i t is l i k e l y to stay, what is needed is a test (or tests) that would differentiate cattle according to the type of Brucella exposure experiences ( i . e . , whether infected with f i e l d strains, vaccinated or non-exposed). The data presented in this study suggests that the in v i t r o LST using antigen+INDO model may have significant diagnostic application in bovine brucellosis.
Further studies should be conducted where a
large number of Brucella exposed cattle w i l l be used to strengthen our findings. ACKNOWLEDGEMENTS Supported in part by the U.S. Department of A g r i c u l t u r e , Veterinary Services and Science Education Administration, and the Minnesota A g r i c u l t u r a l Experiment Station. We thank Diane Klausner for her technical help.
REFERENCES Ferraris, V.A. and Derubertis, F.R., 1974. Release of prostaglandin by mitogen-induced-stimulated leukocytes in culture. J. Clin. Invest. 54 : 378-386. Goodwin, J.S., Bankhurst, A.D. and Messner, R.P., 1977a. Suppression of human T-cell mitogenesis by prostaglandin: evidence of a prostaglandin-producing suppressor c e l l . J. Exp. Med. 146 : 1719-1734. Goodwin, J.S., Messner, R.P., Burkhurst, A.D., Peake, G.T., Saiki, J.H. and Williams, J r . , R.C., 1977b. N. Engl. J. Med. 297 : 963-968. Goodwin, J.S., Messner, R.P. and Peake, G.T., 1978. Prostaglandin suppression of mitogen-stimulated lymphocytes in v i t r o . Changes with mitogen dose and preincubation. J. Clin. Invest. 62 : 753-760. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K., Angus, R.D., Pietz, D.E. and Muscoplat, C.C., 1978a. Kinetics of in v i t r o bovine lymphocyte immunostimulation with a Brucella abortus antigen. Am. J. Vet. Res. 39(2) : 235-239. Kaneene, J.M.B., ~ D.W., Anderson, R.K., Angus, R.D., Pietz, D.E. and Muscoplat, C.C., 1978b. Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19. Am. J. Vet. Res. 39(4) : 585-589. Kaneene, J.M.B., Anderson, R.K., Johnson, D.W., Muscoplat, C.C., Nicoletti, P., Angus, R.D., Pietz, D.E. and Klausner, D.J., 1978c. Whole-blood lymphocyte stimulation assay for measurement of cell-mediated immune responses in bovine brucellosis. J. Clin. Microbiol. 7 : 550-557. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K. and Muscoplat, C.C., 1978d. Comparison of s e n s i t i v i t y and s p e c i f i c i t y of purified lymphocytes and wholeblood in v i t r o lymphocyte stimulation assays in detection of Brucella a b o r t u - s - i ~ i o n in cattle. J. Clin. Microbiol. 8 : 396-40T. Kaneene, J.M.B., Anderson, R.K., Johnson, D.W. and Muscoplat, C.C., 1978e. Brucella antigen preparations for in v i t r o lymphocyte immunostimulation assays in bovine brucellosis. Infect. and Immun. 22(2) : 486-491. Kaneene, J.M.B., Johnson, D.W., Anderson, R.K. and Muscoplat, C.C., 1978f. U t i l i z a t i o n of a specific in v i t r o immunostimulation assay as an aid in detection of B r u c e l l a - i n f e ~ e ~ t l e not detected by serological tests. J. Clin. Microbiol. 8 : 512-515.
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Kaneene, J.M.B., Anderson, R.K., Johnson, D.W., Angus, R.D., Muscoplat, C.C., Pietz, D.E., Vanderwagon, L.C. and Sloane, E.E., 1978g. Responses in swine from a herd infected with Brucella suis. Am. J. Vet. Res. 39(10) : 1607-1611. Mihas, A.A., Gibson, R.G. and Hirschowitz, B . I . , 1975. Suppression of lymphocyte transformation by lb. (16) diamethyl prostaglandin E2 and unsaturated f a t t y acids. Proc. Soc. Exp. Biol. Med. 149 : 1026-1028. Muscoplat, C.C., Rakich, P.M., Thoen, C.O. and Johnson, D.W., 1978. Enhancement of lymphocyte blastogenic and delayed h y p e r s e n s i t i v i t y skin responses by indomethacin. Infect. and Immun. 20 : 627-629. Webb, D.R., Nowowiejski, I . , Dauphinne, M. and Talal, N., 1977. Antigeninduced alterations in splenic prostaglandin and cyclic nucleotiole levels in N2B mice. J. Immunol. 118 : 446-450. Webb, D.R., Rogers, T.I. and Nowowiejski, I . , 1979. Endogeneous prostaglandin synthesis and the control of lymphocyte function. Ann. NY. Acad. Sci. 332 : 262-270. Zimecki, M. and Webb, D.R°, 1976. The regulation of the immune response to Tindependent antigens by prostaglandins and B-cells. J. Immunol. 117 : 2158-2164.