Effect of erythromycin on the immune response and interferon production

lmmunopharmacology, 14 (1987) 101-106

Elsevier

101

IMO 00383

Effect of erythromycin on the immune response and interferon production A l b e r t o Biglino 1, B r u n e l l a F o r n o 1, A n n a m a r i a P o l l o n o 1, M a r g h e r i t a Busso 1, M i c h e l a M a s c o l o 1, A g o s t i n o Pugliese 1, P i e r a n g e l o T o v o z a n d P a o l o G i o a n n i n i 1 l lnstitute for Infectious Diseases and 2Institute of Paediatric Clinic, University of Torino, Torino, Baly

(Received 12 March 1987; accepted 7 July 1987)

Abstract: The influence of erythromycin on some aspects of humoral and cell-mediatedimmunity has been examined employinghuman as well as murine models, both in vivo and in vitro. No significant differencesin antibody synthesis, ~- and 7-interferon yield, cutaneous delayed hypersensitivityor lymphocyteblastogenic response to mitogens have been detected between erythromycin-treatedsubjects and controls. Similarly,in vitro tests on interferon production and blastogenic response to mitogens showed no significant differenceswhen performed with and without erythromycin. Therefore, in contrast with many other antibacterial drugs, erythromycin seems to be devoid of any adverse effects on the immune system. Key words: Antibiotics; Erythromycin; Immunity; Interferon

Introduction

Everyday medical practice often requires antibiotic prescription to outpatients on empirical bases. This is particularly true in such situations as acute respiratory tract infections, where a viral or bacterial aetiology often remains undefined. Antibiotics are also frequently employed in severe viral infections, such as lower respiratory tract disease caused by influenza viruses, with the aim to prevent bacterial complications. In both cases the integrity of the host's immune system is the basic requisite for the antibiotic to achieve a clinical result. Nevertheless, a lot of evidence is accumulating on the ability by chemotherapeutic drugs to interfere with structures and metabolic steps playing a Correspondence." A. Biglino, Istituto di Malattie Infettive, Corso Svizzera 164, 10149 Torino, Italy. Abbreviations: Con-A, concanavatin A; NDV, Newcastle disease virus; PBS, phosphate buffered saline; PHA, phytohaemagglutinin; PWM, pokeweed mitogen; SRBC, sheep red blood cells.

key role in protein and nucleic acid synthesis not only of the prokaryotic, but also of the eukaryotic cell (Gillissen, 1985). Indeed, recent studies suggest that some widely employed antibiotics, such as tetracyclines, aminoglycosides, rifampin and chloramphenicol are able to modify the immune function in several ways (Hauser et al., 1982). Although reports on negative effects predominate in the literature, concerning mainly chemotaxis, lymphocyte transformation and delayed hypersensitivity (Hauser et al., 1982), some enhancing activities by phosphomycin on humoral immunity (Gillissen, 1986) and by erythromycin on phagocytes protein synthesis (Forsgren, 1984) have been described. Quite strangely, these studies only rarely focused on macrolides, a class of substances often employed as first-line antibiotics in acute respiratory tract diseases, because of their relative lack of side effects coupled with efficacy and low cost. To be considered is the fact that their impact on cell-mediated immunity and anti-viral defence mechanisms is far

102 from having been extensively investigated. Among these mechanisms, the interferon system plays a fundamental role in controlling viral infections (Merigan et al., 1982), both directly through its peculiar anti-viral activity, as well as indirectly, by modifying the host's immune reactivity (Stiehm, 1982); nevertheless, only limited information is available on the possible interactions between chemotherapeutic drugs and interferon synthesis and release (Rollag, 1976; Ustacelebi et al., 1972; Pugliese et al., 1981). Furthermore, a nearly total lack of information exists, to our knowledge, about the interactions between macrolides and the interferon system. For these reasons, in the present study we have assessed the effects of erythromycin on some aspects of humoral and cell-mediated immunity, both in vivo and in vitro, with particular emphasis on ~- and 7-interferon production.

Materials and Methods

In vivo studies Effects on humoral immune response in mice. Two groups of C3H mice weighing 23 g (mean) were treated intraperitoneally with, respectively, 30 mg/kg and 150 mg/kg of erythromycin lactobionate in a volume of 100/d. A third group of C3H mice was treated in the same way with buffered saline only. One hour apart, 1 x 108 sheep red blood cells (SRBC) were administered intraperitoneally to all animals of the three groups. Six days later all mice were bled to death. Blood from each mouse was collected and, after clotting, serum was inactivated at 56°C for 20 min. Haemagglutinating antibodies were titrated as described elsewhere (Murray et al., 1974). Briefly, 25/tl of a 2% suspension of SRBC in veronal buffer, pH 7.2, were added to 25/A of each serum sample, diluted with the same buffer at a ratio of 2 in a round-bottom, 96-well microtiter plate. Haemagglutinating titre was observed after overnight incubation at + 4°C.

Effects on cell-mediated immune re,sponse in mice and human volunteers. Contact sensitivity was induced in three groups of C3H mice weighing 22 g (mean), treated with 30 and 150 mg/kg erythromycin lactobionate, and with plain buffered saline, respectively, as described above, by painting the shaved skin of the abdomen with 100/d of a 2% ethanolic solution of oxazolone (Asherson et al., 1968). The development of sensitization was assessed one week later by applying on both ears of all animals 100/d of a 1% solution of oxazolone in olive oil. Ear thickness was measured before treatment and 48 h later by means of a 'Panter' micrometer. In order to study the effects of erythromycin on human delayed-type hypersensitivity, thirty outpatients seeking medical care because of uncomplicated upper respiratory tract disease were randomly allocated to a control group, treated with symptomatic drugs, or to a treated group taking symptomatics plus erythromycin (2 g/day orally). The control group included 9 males and 6 females (mean age 35 ± 8 years) while the treated group included 8 males and 7 females (mean age 31 + 9 years). All patients reported routine immunization with tetanus and diphtheria toxoids. After five days of treatment all patients underwent a multi-puncture intradermal test (Multitest C.M.I., Merieux) including seven T-dependent antigens (PPD, tetanus and diphtheria toxoids, Candida, Trycophyton, Proteus and Streptococcus antigens) and a glycerol control, in order to assess the ability to recognize antigens and to mount a delayed-type hypersensitivity response. Results were expressed as a score obtained by the sum (in ram) of the mean infiltration diametres after 48 h (Kniker et al., 1979). In vitro studies Effects on human lymphocyte transformation by mitogens. Peripheral blood mononuclear cells were obtained from four healthy donors (2 males, 2 females; mean age 25 ± 5 years) by centrifugation on Ficoll Hypaque, washed thrice in phosphate buffered saline (PBS) and resuspended at a concentration of 2 × 106/ml in RPMI-1640 medium (Flow

103 Laboratories) containing 10% foetal calf serum (Flow), 2 mM glutamine and 2 g/1 sodium bicarbonate (complete medium). Cells from each donor were stimulated in triplicate in a 96-well, flat-bottom microtiter plate (NUNC) with phytohaemagglutinin (PHA; Wellcome), concanavalin-A (ConA; Sigma) and pokeweed mitogen (PWM; Flow) at final concentrations of 20, 12.5 and 20/~g/ml, respectively, while three control wells contained medium only. Culture medium in the first five rows of each plate contained erythromycin succinate at a final concentration of 1, 0.5, 0.1, 0.01 and 0.001 mg/ml, respectively, while the sixth row did not contain the antibiotic. After three days, cultures were pulsed for 18 h with [3H]thymidine (1 /~Ci/ well; Sorin Biomedica, Italy) and then each culture was harvested with a semiautomated device (Skatron; Flow) on fiberglass disks. The amount of incorporated radioactivity was measured by a liquid scintillation system (Rackbeta; LKB-Wallac) and expressed as the difference between mean counts per minute with mitogen and mean counts per minute without mitogen (A CPM). Effects on ct- and 7-interferon production. Peripheral blood mononuclear cells were obtained from each of the above-mentioned donors as described, resuspended at a concentration of 1 x 106/ml in complete medium and stimulated with Newcastle disease virus, strain F (NDV-F) at a multiplicity of 100:1, or with PHA-M (Wellcome) at a final concentration of 40/~g/ml, or with medium only. Six sets of such cultures were set up from each donor, the first five containing erythromycin base at a final concentration of 1, 0.5, 0.1, 0.01 and 0.001 mg/ml, respectively while the sixth one did not contain the antibiotic. After 24 h for thd NDV-stimulated cultures, and 72 h for the PHAstimulated ones, the supernatants were collected and induced interferons titrated on WISH cell monolayers in microtiter plates, employing vesicular stomatitis virus at a multiplicity of 1:1 as challenge, as described elsewhere (Pugliese et al., 1985). Interferon levels were calculated as the reciprocal of the dilution protecting 50% of cell layer from cytopathogenic effect. A working standard of

human 7-IFN equivalent to 64 units was included in each titration. Effects of oral treatment on lymphocyte transformation tests and interferon production. A 30-ml venous blood sample was collected from each volunteer (from both groups, treated and not treated; see section on cell-mediated immune respone), and mononuclear cells, after separation, were stimulated with PHA, Con-A or PWM as already described, in order to evaluate lymphocyte transformation ability, and with NDV, and PHA at a concentration of 40 #g/ml, in order to assess ~- and 7-interferon production, respectively, as described above.

Results

In vivo studies (a) A slight but not significant increase in haemagglutinin production against SRBC could be detected in erythromycin-treated mice if compared to controls at both the employed doses of 30 and ! 50 mg/kg body weight (Fig. 1.). (b) No difference in delayed response to cutaneous challenge with oxazolone became evident between

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Fig. 1. Haemagglutinatingtitre (log2) against SRBC (open bars) and ear thickness increase (cm x 10-3; shaded bars) after oxazolone challenge, in C3H mice treated with 30 or 150 mg/kg erythromycinor buffered saline only (controls).

104 ieux) did not show any significant difference between treated and non-treated volunteers. Scores of 20.9 + 7.02 and 23.6 + 8 were evidenced in patients taking erythromycin or symptomatic drugs, respectively. The first group reacted to 3.02 ± 1.7 antigens, while the second reacted to 3.8 + 1.8 (mean values) (Fig. 2).

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Fig. 2. Mean multitest score (in mm) and number of positive antigens in patients taking erythromycin (2 g/day orally; open bars) and in controls (shaded bars).

non-treated mice, and those treated intraperitoneally with 30 or 150 mg/kg erythromycin. Indeed, increase in ear thickness (mean) was 6.4 x 10 3 cm in the first, 6.42 x 10 3 cm in the second, and 6.45 x 10 -3 cm in the third group (Fig. 1). Skin tests to ubiquitous antigens (Multitest, Met-

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Fig. 3. Effects of different concentrations of erythromycin in vitro on lymphocyte transformation in response to PHA (m), Con-A ( x ) , PWM ( + ) and medium only ([5]).

In vitro studies

( a ) No significant differences in blastogenic response to the employed mitogens (PHA, Con-A and PWM) could be evidenced in vitro between cultures performed in the presence of different concentrations of erythromycin and those performed in its absence (Fig. 3). A slight (though not significant) increase in response was detected in cultures performed with erythromycin concentrations similar to those obtained in vivo after oral administration (1 to 10 /~g/ml) (Goodman). An even better response to P H A was obtained at higher antibiotic concentrations (100 to 500 /~g/ml) (though these concentrations are never attained in vivo). (b) No significant differences in :¢- or 7-IFN production in vitro could be evidenced between lymphocytes cultured in the presence of different erythromycin concentrations or in its absence (Fig. 4). It is to be noted that a slight increase in 7-IFN yield (not statistically significant) was obtained in the presence of antibiotic concentrations very near to the therapeutical ones.

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(c) A satisfying response by peripheral blood mononuclear cells from both treated and non-treated patients was obtained after mitogen stimulation. Mean A-CPM values of 50238 4- 20400 and 55228 ± 31690 with PHA, 33832 ± 17500 and 33451 ± 24700 with Con-A, and 12543 + 8400 and 11088 + 10270 with PWM were obtained respectively from erythromycin-treated and non-treated patients (Fig. 5). The same was true when ct- and ),-IFN production was considered. Mean yields of 7.4 + 1.23 and 7.93 + 0.9 log IU 7-IFN, and 7.27 ± 1.53 and 7.93 + 1.4 log IU ~-IFN were obtained, respectively, from treated and non-treated patients (Fig. 6).

Discussion

The present study demonstrates that no adverse effects of erythromycin on immune reactivity could be detected employing different experimental human or murine models, both in vivo as well as in vitro. Among the other intriguing results, concerning many functional aspects of humoral and cell-mediated immunity, the lack of any negative influence on interferon production should be particularly stressed. This peculiar finding is even more important when the role played by interferons in acute viral infections is considered, taking into account that in respiratory tract diseases antibiotic therapy is often prescribed on empirical bases, without a

precise knowledge of the viral or bacterial aetiology. Erythromycin seems to be devoid of negative influences on interferon producton not only in vivo, but also in vitro, where concentrations as high as 1 mg/ml were attained without impairing ~- or 7IFN yield. Both of these substances are equally important in defence against viral infections, the first one being produced directly by infected leukocytes, and acting as a first-line inhibitor of viral spread, and the second being released by activated T-lymphocytes together with interleukin-2 (Perussia et al., 1980; Schrober et al., 1984) in order to enhance natural-killer (Trinchieri et al., 1978) and T-cytotoxic cell activity against infected cells (Farrar et al., 1981). A total lack of interference on T and B cell replication and DNA synthesis in response to mitogens has been demonstrated after oral administration of erythromycin to volunteers, as well as after in vitro challenge of donors' lymphocytes with a wide range of antibiotic concentrations. This finding clearly demonstrates that erythromycin, even at concentrations one thousand times greater than those obtained after oral administration, is by no means able to modify significantly the ability of immunocompetent cells to proliferate, and thus to respond with a rapid expansion towards an infectious challenge. The integrity of the mechanisms involved not only in lymphocyte proliferation, but also in antigen recognition (which is known to involve antigen processing by macrophages, interleukin-1 release and antigen presentation to lymphocytes; Lesourd et al., 1982) is well evidenced by murine and human in vivo studies, where oxazolone and a panel of ubiquitous T-dependent antigens (Multitest, Merieux) were recognized, respectively, by treated and non-treated subjects without any significant difference, even at doses as high as 150 mg/kg (in the mouse). Finally, in our study antibody synthesis did not seem to be negatively affected even being slightly enhanced - - by intraperitoneally administered erythromycin in the mouse, although the difference in haemagglutinating titres between control and treated mice was not significant. In conclusion, our

106

data allow us to affirm that erythromycin is completely devoid of any adverse effect on antibody synthesis, interferon production, proliferative ability of immunocompetent cells and delayed-type hypersensitivity response. For these reasons it could be considered as a first-choice antibiotic in clinical situations such as acute respiratory tract diseases, where viral and bacterial pathogens are often together in action, and the integrity of both humoral and cell-mediated immune mechanisms must be respected from the beginning of treatment.

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