The influence of antibiotics on the immune system. II. Modulation of fish leukocyte responses in culture

Veterinary Immunology and Irnmunopathology, 9 (1985) 251--260 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

251

THE INFLUENCEOF ANTIBIOTICS ON THE IMMUNESYSTEM, II. MODULATIONOF FISH LEUKOCYTE RESPONSESIN CULTURE J.L. GRONDEL1'2 A.G.M. GLOUDEMANS1 and W.B. VAN MUISWINKEL1 Department of Experimental Animal Morphology & Cell Biology (1) and Department of Animal Husbandry(2), Agricultural University, P.O. Box 338, 6700 AH Wageningen, The Netherlands. (Accepted 7 February 1985) ABSTRACT Grondel, J.L., Gloudemans, A.G.M. and van Muiswinkel, W.B., 1985. The influence of antibiotics on the immune system. II. Modulation of fish leukocyte responses in culture. Vet. Immunol. Immunopathol., 9: 251-260. We report the immunomedulating effect of two tetracycline analogues (oxytetracycline and doxycycline) on mitogenic and allogeneic stimulation of carp (Cyprinus oarpio) leukocytes in vitro. Both drugs interfered with 3H-thymidine incorporation into the DNA of phytohaemagglutinin-stimulated pronephric leukocytes in a dose-dependent manner. Low concentrations of oxytetracycline delayed the mitogenic response, but did not reduce i t . In addition, several other antibiotics were tested for their possible interference with blastogenesis. INTRODUCTION

Since the f i r s t isolation of tetracycline from Streptomyces aureofacien8 in 1948 much work has been done on side effects of these drugs. Impaired phagocytic function of human leukocytes was observed after in vitro addition of chlor.tetracycline (Munozand Geister, 1950). Interference with immunological defence mechanisms was caused by several analogues, including tetracycline (TC), doxycycline (DC), lymecycline (LC) and minocycline (MC). Incubation of human neutrophils with TC or DC caused a decreased capacity to phagocytize yeast and bacteria. Leukocytes harvested from healthy donors after ingestion of TC also demonstrated a decreased phagocytic capacity for yeast (Forsgren et al., 1974). In rats, i t was shown that TC pretreatment had a suppressive effect on carbon clearance (Altura et a l . , 1966). One of the properties of neutrophils is the directed migration induced by external stimuli. Both spontaneous and induced migration of human leukocytes in vitro was severely depressed by LC and DC (Belsheim et a l . , 1979). Corresponding results were obtained in experiments on healthy volunteers given recommended dosages of the antibiotics and in patients treated with DC for post-operative infections. TC also inhibited the migration of human leukocytes in vitro (Forsgren and Schmeling, 1977; Forsgren et a l . , 1978). In addition, mitogenic responses of human T and B lymphocytes were adversely affected by several tetracycline ana0165-2427/85/$03.30

© 1 9 8 5 E l s e v ~ r S c ~ n c e Publishers B.V.

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logues (Banck and Forsgren, 1979; Thong and Ferrante, 1979). A n t i b i o t i c immunomodulation is not r e s t r i c t e d to the t e t r a c y c l i n e s . The wide range of a n t i b i o t i c s which modulate immune responses i n v i t r o as well as i n vivo have been reviewed (Finch, 1980; Hauser and Remington, 1982). However, reports dealing with antimicrobial agents a f f e c t i n g the immune system of f i s h are scarce. Oxytetracycline (OxyTC) severely reduced the in vivo immune reponse of carp, evidenced by the prolongation of scale a l l o g r a f t survival and by suppression of the humoral response to sheep red blood c e l l s (Rijkers et a l . ,

1980,

1981). Immunosuppression has also been demonstrated in rainbow t r o u t a f t e r feeding with p e l l e t s containing OxyTC (Anderson et al.~ 1984; van Muiswinkel et al.,

1984). In a study designed to determine i f a n t i b i o t i c s might d i r e c t l y i n -

fluence immunocytes i n v i t r o , we found that OxyTC i n h i b i t e d mitogenic responses of carp leukocytes (Grondel and Boesten, 1982). Since OxyTC has widespread therapeutic applications in f i s h c u l t u r e , i t is important to understand the d e t a i l s of i t s immunomodulating e f f e c t s . We have now done f u r t h e r work to obt a i n more i n s i g h t into the mode of action of t h i s drug, and have tested several other a n t i b i o t i c s for t h e i r possible interference with blastogenesis.

MATERIALS AND METHODS Animals Carp (Cyprin~s oarpio L . ) , 6-8 months of age, were bred in our laboratory and kept in aquaria with aerated running tapwater at a temperature of 22o + 1°C. Animals were fed d a i l y with pelleted dry food (K30, Trouw & Co., Putten, The Netherlands) by means of a "Scharflinger" automatic feeder. Antibiotics The f o l l o w i n g i n j e c t a b l e a n t i b i o t i c s were used: o x y t e t r a c y c l i n e (Engemycine~ Mycofarm B.V., De B i l t ,

The Netherlands), doxycycline (Vibramycin~ P f i z e r B.V.,

Rotterdam, The Netherlands), a combination of sulfatroxazole and trimethoprim ( r a t i o 5:1, Leotrox~ Leo B.V., Emmen, The Netherlands), sulfadimethoxine ( I n t e r v e t B.V., Boxmeer, The Netherlands), a combination of lincomycin and spectinomycin ( r a t i o 1:2, Linco-Spectin~ Upjohn, Ede, The Netherlands), gentamicin (Gentocin-P~ A.C.F. Chemiefarma N.V., Maarssen, The Netherlands), a m p i c i l l i n (Penbritin~ Beecham Farma B.V., Maarssen, The Netherlands), and chloramphenicol ( I n t e r v e t B.V.). Powdered forms of sulfadimidine (Aesculaap B.V., Boxtel, The Netherlands), lincomycin-spectinomycin ( r a t i o 1:2, Upjohn) and furaltadone (Furaltadone~ A.O.V. Cuyk, The Netherlands) were used. The solvent of the oxyt e t r a c y c l i n e s o l u t i o n was k i n d l y provided by Gist-Brocades, D e l f t , The Netherlands. The commercially available a n t i b i o t i c s were f r e s h l y dissolved in

253

RPMI-1640 and added once at the s t a r t of the cultures. Cell cultures Standard RPMI-1640 culture medium (Flow, I r v i n e , Scotland) was buffered with 2.1 g/ml NaHCO3 (pH 7.4), and supplemented with 2 mM L-glutamine (Merck, Darmstadt, F.R.G.)° and 10% heat-inactivated pooled carp serum (PCS). Pronephric leukocytes were prepared and cultured as described before (Grondel and Boesten, 1982; Grondel and Harmsen, 1984). In b r i e f , single cell suspensions were obtained by teasing the tissue through a nylon sieve, washed twice and f i n a l l y resuspended in serum free culture medium. I n ~ l l

experiments cell v i a b i l i t y was

assessed using the trypan blue exclusion method, Cells were cultured for four days (unless otherwise specified) in round bottom m i c r o t i t e r plates (M24, Greiner, NUrtingen, F.R.G.) in a final volume of 0.2 ml per well in the presence of phytohaemagglutinin (50 ~g/ml, PHA-P, Difco, Detroit, U.S.A.) or in medium only. Two-way mixed leukocyte cultures were performed with peripheral blood leukocytes according to the method described by Gloudemans and Cohen (in prep.). Leukocytes were p u r i f i e d from hepariniz~d blood by Percoll (Pharmacia, Uppsala, Sweden) cushion centrifugation. Cells collected from the interface were washed twice and f i n a l l y resuspended in medium supplemented with 50 uM mercaptoethanol (Merck) and 0.2% PCS. Peripheral blood leukocytes (2.5 x 105 ) from each animal were cultured for 6 days in round-bottom m i c r o t i t e r plates under conditions identical to the proneph~ic leukocyte cultures mentioned above. In these experiments the individual variation was about 25%. Measurement of DNA synthesis DNA synthesis was assayed by 3H-thymidine (3H-TdR) incorporation a f t e r a 16-h

pulse with 0.4 uCi 3H-TdR (s.a. 5 Ci/mMol, Radiochemical Centre, Amersham,

England) per well. Cells were harvested onto g l a s s - f i b e r f i l t e r s

(Bioproducts,

Walkersville, U.S.A.) using a multiple automated sample harvester (Mash I I , Dynatech, NUrtingen, F.R.G.). The f i l t e r s were dried and r a d i o a c t i v i t y was determined by s c i n t i l l a t i o n spectrometry. Results are given for 2-4 representative experiments. Results of t r i p l i c a t e or quadruplicate cultures are expressed as the mean c.p.m. ± S.D., unless otherwise stated. The Student's t test was used for s t a t i s t i c a l evaluation.

254 RESULTS Effect of t e t r a c y c l i n e s on the mitogenic response of leukocytes Both OxyTC and DC i n h i b i t e d 3H-thymidine incorporation into the DNA of PHA-stimulated pronephric leukocytes. The effects were dose-dependent (Figure i ) . cpm .

.

.

.

.

10 5 ,

\ -,.\ 10 4 . /

10 3

\°J

•

\ \o-~--~_\~__

\ 10 2

0:15 0..31 0:62 1,75 2:5

;

10

20#g/ml

Fig. 1. The e f f e c t of OxyTC ( I - - I ) , DC ( A - - A ) and the solvent of the OxyTC s o l u t i o n ( 0 ~ 0 ) on PHA-stimulated (closed symbols) and3non-mitogen treated pronephric leukocytes (open symbols). Incorperation of H-TdR in control cultures without a n t i b i o t i c s is indicated with asterisks (~). Cultures were harvested on day 4. Results are expressed as mean c.p.m, observed in t r i p l i c a t e cultures. The S.D. was about 10%. At concentrations of 4-6 pg OxyTC and 1-2.5 ~g DC per ml, the responses were reduced to about 50% of those of the mitogen-stimulated controls. DC was more suppressive than OxyTC. The difference between the two analogues may r e l a t e to the better l i p i d - s o l u b i l i t y

of doxycycline. Addition of Ca2+ in concentrations

up to 1.7 mM to the c u l t u r e medium, to compensate f o r the possible chelation of d i v a l e n t cations by the drugs, did not r e l i e v e the impaired response (results not shown). In order to exclude possible effects caused by the solvent, pronephric leukocytes were also exposed to various concentrations of the OxyTC solvent. Neither the background DNA synthesis of unstimulated control c e l l s nor the same process in mitogen-activated leukocytes was s i g n i f i c a n t l y affected by the solvent (see Figure I ) ,

255

Kinetic change in the mitogenic response PHA-stimulated pronephric leukocytes were cultured in the presence of 5, 10 or 15 ~g OxyTC per ml. Cultures were~harvested 2, 3, 4, 5, 6 or 7 days a f t e r initiation

of the c u l t u r e . 3H-thymidine was added to the cultures 16 hours

before harvesting. Figure 2 demonstrates a dose-dependent delay in the leukocyte response rather than a real suppressive e f f e c t by OxyTC. However, in the OxyTC treated cultures the peak was shifted to day 5 or l a t e r . Obviously, the impairment of c e l l u l a r functions l i k e DNA synthesis is not due to c y t o t o x i c i t y as suggested in previous investigations (Grondel and Boesten, 1982), cpm

10 5

10 4

",ii; ½

½

4

5

6

7days

Fig. 2. Kinetics of thymidine uptake into PHA-stimulated pronephric leukocytes in the presence of 0 (O= c o n t r o l ) , 5 ( * ) , 10 (0) or 15 (D) ~g OxyTC per ml culture medium. Results are expressed as mean c.p.m, observed in quadruplicate cultures. The S.D. was about 10%.

Under our c u l t u r e conditions the i n h i b i t o r y e f f e c t on DNA synthesis was t r a n s i e n t . The background level of 3H-thymidine uptake by unstimulated control c e l l s gradually decreases with time. Effect of t e t r a c y c l i n e s on the mixed leukocyte c u l t u r e In a l l experiments mentioned above, leukocytes were p o l y c l o n a l l y activated by PHA. Stimulation of DNA synthesis without mitogens can be achieved by c u l t u r i n g peripheral blood or pronephric leukocytes from g e n e t i c a l l y d i s s i m i l a r i n d i v i d u a l s (mixed leukocyte c u l t u r e ) . Addition of the t e t r a c y c l i n e analogues to such cultures at concentrations of I0 ~g OxyTC or 5 ug DC per ml also reduced the 3H-thymidine incorporation markedly: 61% and 98% for OxyTC and DC respective l y (Figure 3). The influence of d i f f e r e n t a n t i b i o t i c s on the mitogenic response D i f f e r e n t a n t i b i o t i c s were added in various concentrations to routine PHA s t i m u l a t i o n tests (Figure 4). Five a n t i b i o t i c s

~ulfatroxazole/trimethoprim

(5:1), sulfadimethoxine, sulfadimidine, lincomycin/spectinomycin (1:2, i n j e c table solution) and ampicillin-~ did not suppress the mitogenic response of the pronephric leukocytes. On the contrary, an increased incorporation of 3H-thymidine was observed especially at low concentrations. Gentamicin and furaltadone

256

F i g . 3. The e f f e c t o f OxyTC (10 ~g/ml) or DC (5 pg/ml) on the DNA s y n t h e s i s o f a l l o g e n e i c stimulated peripheral blood leukoc y t e s (PBL). PBL o f g e n e t i c a l l y d i s s i m i l a r i n d i v i d u a l s (A and B) were c u l t u r e d f o r 6 days. The results of quadruplicate culture! are expressed in c . p . m . + S.D.

A control B control

+

A÷B control

A÷B+DC A+ B+OxyTC

~--~ ---~ 103

104

105 c p m

showed a dose-dependent i n h i b i t i o n was s t i m u l a t e d quantities

o f the m i t o g e n i c

by c h l o r a m p h e n i c o l

at concentrations

equal t o or e x c e e d i n g t h i s

concentration

response.

Thymidine uptake

less than 5 ~ g / m l , whereas became s u p p r e s s i v e .

Antibiotic concentralion 01 0.5 1 5 10 20 40~Jg/mt

5o] • 50 J

i

5°7 50]

m~ •

i

5O7 I 5o] .c___5°l ]5 5o]

•

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mm• .. _ I

_ i

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---

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4

50~__~_~_,

+ Sulfadirnidine 2 --i_

+ Lincomycin/spectinomycin 2

i

~, + Chloramphenicoll _

+

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Sulfadimethoxine 1

+ Lincomycin/spectinomycin 1

--

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~ 55~]

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SulfatroXazole/trirnethoprim 1

Ampicillin'

• + Gentamicin 1 = i l l

-

+_ Furaltadone2

lOO F i g . 4. Several a n t i b i o t i c s were t e s t e d f o r t h e i r p o s s i b l e i n t e r f e r e n c e w i t h DNA s y n t h e s i s . P H A - a c t i v a t e d p r o n e p h r i c l e u k o c y t e s were c u l t u r e d f o r 4 days in the absence o r presence o f v a r y i n g c o n c e n t r a t i o n s o f d i f f e r e n t a n t i b i o t i c s . The r e s u l t s o f t r i p l i c a t e c u l t u r e s are e x p r e s s e d in p e r c e n t a g e s t i m u l a t i o n o r inhibition compared w i t h m i t o g e n - t r e a t e d c o n t r o l c u l t u r e s . 1) i n j e c t a b l e , 2) powder and m) not t e s t e d .

257

DISCUSSION Our results show that OxyTC and DC a f f e c t mitogenic as well as allogeneic responses of carp leukocytes i n v i t r o .

This confirms our previous data showing

that the incorporation of 3H-thymidine into DNA of PHA- or LPS-stimulated leukocytes was depressed (Grondel and Boesten, 1982). Furthermore, the results show that OxyTC influences the k i n e t i c s of the response. The difference between the two t e t r a c y c l i n e analogues in t h e i r a b i l i t y to suppress the i n v i t r o

response can be explained by t h e i r chemical structure

and related properties (Neu, 1978). For instance, the chelating properties of t e t r a c y c l i n e s for d i v a l e n t cations could i n t e r f e r e with the mitogenic response of leukocytes (Diamantstein and Odenwald, 1974). Our results are in agreement with those reported by Banck and Forsgren (1979) and Thong and Ferrante (1979) who showed that DC profoundly i n h i b i t e d p r o l i f e r a t i v e responses. At low concentrations DC suppressed both the mitogenic responses of human T and B lymphocytes and i n v i t r o antibody production (Banck and Forsgren, 1979). The r e v e r s i b i l i t y of the i n h i b i t o r y e f f e c t was also studied by these authors: preincubation of the c e l l s for i h in i0 ug DC per ml culture

medium supplemented with 10% serum did not a f f e c t

mitogenic responsiveness during subsequent c u l t u r i n g in the absence of t e t r a cyclines (Thong and Ferrante, 1979). However, lymphocytes could not be restimulated when washed a f t e r two days of incubation with 50 ~g DC per ml serum-free c u l t u r e medium (Banck and Forsgren, 1979). The i r r e v e r s i b l e e f f e c t of t h i s high drug concentration may be due to i n h i b i t i o n of cytoplasmatic protein synthesis (Banck and Forsgren, 1979; Gijzel and Kroon, 1978). At low doses (I0 ug/ml), only mitochondrial protein synthesis appears to be i n h i b i t e d (van den Bogert and Kroon, 1981). Thus, the discrepancy mentioned above can be explained by the d i f f e r e n t experimental conditions in these studies. I t should be emphasized that the active drug concentration w i l l decrease with time a f t e r i n i t i a l

addition to the culture. Therefore, the immunosuppres-

sive e f f e c t should be more s t r i k i n g i f there were compensation for the loss of biological a c t i v i t y of the drug throughout the experiment. In addition, our experiments showed that the kinetics of mitogenic responses were changed at low concentrations of OxyTC. In fact the suppression of the 3H-TdR incorporation into DNA was transient. S i m i l a r l y , immune responses may be delayed in vivo: treatment of carp with OxyTC affected the kinetics of the humoral response against sheep red blood c e l l s (manuscript in preparation). Chemotaxis and subsequent phagocytosis are important processes by which pathogenic microorganisms can be rendered inoffensive. I t has been demonstrated that t e t r a c y c l i n e s exert an adverse e f f e c t on these defence mechanisms. This

258

was observed in man, both in vitro (Forsgren et al., 1974; Belsheim etal., 1979; Forsgren and Schmeling, 1977; Forsgren etal., 1978) and in vivo (Forsgren

etal.,

1974; Belsheim et a~., 1979). Tetracyclines are b a c t e r i o s t a t i c agents

and i n t e r f e r e with bacterial protein synthesis at the 30S ribosomal l e v e l . The binding of t e t r a c y c l i n e s to the ribosomes is r e v e r s i b l e . Under appropriate conditions the microorganism can subsequently s t a r t growing again i f the drug leaks out of the bacterium (Neu, 1978). Therefore, impairment of the immune system by b a c t e r i o s t a t i c compounds may have serious implications f o r the outcome of therapy, e s p e c i a l l y when recovery needs a r e l a t i v e l y long period. Interactions between macrophages and lymphocytes are essential f o r the i n duction of a proper immune reponse. Macrophages have a v a r i e t y of functions i n c l u d i n g uptake of foreign material (antigens), degradation and subsequent presentation of antigens to lymphocytes (Unanue, 1980). I t is obvious that impairment of the i n i t i a l

processes in the immune response w i l l decrease the

effectiveness of the defence mechanisms. I t has been demonstrated that macrophages accumulate a wide range of a n t i b i o t i c s , t e t r a c y c l i n e included, when incubated in the presence of drug concentrations s i m i l a r to c l i n i c a l l y appropriate serum levels (Johnson e t a l . ,

1980). As mentioned e a r l i e r , chemo-

t a c t i c and phagocytic processes can be affected by a n t i b i o t i c s .

Investigations

on the production of lymphokines revealed that the secretion of migration i n h i b i t i o n f a c t o r was suppressed by a n t i b i o t i c s , e.g. r i f a m p i c i n (Serrou, 1974). In conclusion, o x y t e t r a c y c l i n e and doxycycline i n t e r f e r e with the mitogenic and allogeneic response of carp leukocytes. Furaltadone, gentamicin and to a lesser extent chloramphenicol and lincomycin/spectinomycin also exerted suppressive effects when tested in the same system. I t is i n t e r e s t i n g that the other tested a n t i b i o t i c s ( s u l f a t r o x a z o l e / t r i m e t h o p r i m , sulfadimethoxine, s u l f a dimidine, i n j e c t a b l e lincomycin/spectinomycin and a m p i c i l l i n ) enhanced the uptake of 3H-TdR. Clearly, the leukocyte s t i m u l a t i o n assay provides a s e n s i t i v e tool for screening side effects of a n t i b i o t i c s .

The antimicrobial agents which

have been found to be responsible for immunomodulation in vitro need f u r t h e r i n v e s t i g a t i o n . However, i t is beyond the scope of t h i s study to subject a l l these compounds and solvents to detailed research. These observations stress the notion that a careful choice has to be made before certain drugs are used. The prolonged a p p l i c a t i o n of t e t r a c y c l i n e s mixed with food, as is usual in some f i s h culture systems f o r the prevention of diseases, should be avoided. On the other hand, these immunomodulating agents may be valuable tools for studying regulation of the immune response in f i s h .

259 ACKNOWLEDGEMENTS We would l i k e to thank Mrs. M.S.J. van der Wal - van Beckum f o r her s k i l f u l

secretarial assistance and Mr. W.J.A. Valen for drawing the i l l u s t r a t i o n s . REFERENCES A l t u r a , B.M., Hershey, S.G., A l i , M. and Thaw, C., 1966. Influence of t e t r a cycline on phagocytosis, i n f e c t i o n and resistance to experimental shock: r e l a t i o n s h i p to m i c r o c i r c u l a t i o n . J. Reticuloendoth. Soc., 3: 447-457. Anderson, D.P., Muiswinkel van, W.B. and Roberson, B.S., 1984. Effects of chemic a l l y induced immune modulation on i n f e c t i o u s diseases of f i s h . In: M. Kende, J. Gainer and M. Chirigos ( e d i t o r s ) , Chemical regulation of immunity in Veterinary Medicine. A.R. Liss I n c . , New York, pp. 187-211. Banck, G. and Forsgren, A., 1979. A n t i b i o t i c s and suppression of lymphocyte function i n v i t r o . Antimicrob. Agents and Chemother., 16: 554-560. Belsheim, J . , Gnarpe, H. and Persson, S., 1979. Tetracyclines and host defense mechanisms: interference with leukocyte chemotaxis. Scand. J. I n f e c t . Dis., 11: 141-145. Bogert van den, J.J. and Kroon, A.M., 1981. Tissue d i s t r i b u t i o n and effects on mitochondrial protein synthesis of tetracyclines a f t e r prolonged continuous intravenous administration to rats. Biochem. Pharmacol, 30: 1706-1709. Diamantstein, T. and Odenwald, M.V., 1974. Control of the immune response i n v i t r o by calcium ions. I. The antagonistic actions of calcium ions on cell p r o l i f e r a t i o n and on cell d i f f e r e n t i a t i o n . Immunology 27: 531-541. Finch, Ro, 1980. Immunomodulating effects of antimicrobial agents. J. A n t i microb. Chemother., 6: 691-699. Forsgren, A., Schmeling, D~ and Quie, P.G., 1974. Effect of t e t r a c y c l i n e on the phagocytic function of human leukocytes. J. I n f e c t . Dis., 130: 412-415. Forsgren, A. and Schmeling, D., 1977. Effect of a n t i b i o t i c s on chemotaxis of human leukocytes. Antimicrob. Agents Chemother., 11: 580-584. Forsgren, A., Schmeling, D. and Banck, G., 1978. Effect of a n t i b i o t i c s on chemot a x i s of human polymorphonuclear leukocytes in vitro. I n f e c t i o n 6 (suppl. I ) : 8102-8106. G i j z e l , W.P. and Kroon, A.M., 1978. On the protection of bone marrow against t o x i c side effects of t e t r a c y c l i n e s . J. Mol. Med., 3: 157-165. Grondel, J.L. and Boesten, H.J.A.M., 1982. The influence of a n t i b i o t i c s on the immune system I. I n h i b i t i o n of the mitogenic leukocyte response i n v i t r o by o x y t e t r a c y c l i n e . Dev. Comp. Immunol., Suppl. 2, pp. 211-216. Grondel, J.L. and Harmsen, E.G.M., 1984. Phylogeny of i n t e r l e u k i n s : growth factors produced by leukocytes of the c y p r i n i d f i s h , Cyprinus carpio L. Immunology 52: 477-482. Hauser, W.E. and Remington, J.S., 1982. Effects of a n t i b i o t i c s on the immune response. Am. J. Medicine, 72: 711-716. Hesketh, T.R., Smith, G.A., Houslay, M.D., Warren, G.B. and Metcalfe, J.C., 197~ Is an early calcium flux necessary to stimulate lymphocytes? Nature, 267: 490-494. • Johnson, J.D., Hand, W.L., Francis, J.B., King-Thompson, N. and Corwin, R.W., 1980. Antibiotic uptake by alveolar macrophages. J. Lab. Clin. Med., 95: 429-439. Munoz, J. and Geister, R., 1950. Inhibition of phagocytosis by aureomycin. Proc. Soc. Exp. Biol. Med., 75: 367-370. Muiswinkel van, W.B., Anderson, D.P., Lamers, C.H.J., Egberts, E., Loon van, J.J.A. and IJssel, J.P., 1984. Fish immunology and fish health. Fish Immunology, M.J. Manning & M.F. Tatner, eds., Proceedings of the Plymouth Meeting 1983, Academic Press, London (in press). Neu, H.C., 1978. A symposium on the tetracyclines: A major appraisal. Bull. N.Y. Acad. Med., 54: 141-155.

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Rijkers, G.T., Teunissen, A.G., Oosterom van, R. and Muiswinkel van, W.B., 1980. The immune system of c y p r i n i d f i s h . The immunosuppressive e f f e c t of the a n t i b i o t i c o x y t e t r a c y c l i n e in carp. Aquaculture, 19: 177-189. Rijkers, G.T., Oosterom van, R. and Muiswinkel van, W.B., 1981. The immune system of c y p r i n i d f i s h . Oxytetracycline and the regulation of humoral immunity in carp. Vet. Immunol. Immunophathol., 2: 281-290. Serrou, B., 1974. Rifampicin and immunosuppression. Lancet, 11: 172. Thong, Y.H. and Ferrante, A., 1979. I n h i b i t i o n of mitogen-induced human lymphocyte p r o l i f e r a t i v e responses by t e t r a c y c l i n e analogues. Clin. Exp. Immunol., 35: 443-446. Unanue, E.R., 1980. Cooperation between mononuclear phagocytes and lymphocytes in immunity. New England J. Med., 303: 977-985.