Clin. exp. lmmuMI. (1979} 35,443-446.
`
`Inhibition of mitogen-induced human lymphocyte proliferative
`responses by tetracycline analogues
`
`Y, H. THONG & A. FERRANTE Department of Paediatrics, The University of Adelaide, The Adelaide Children's
`Hospital, North Adelaide, 5006 South Australia
`
`(Recrived 20 December 1977}
`
`SUMMARY
`The effect of tetracyclines on mitogen-induced proliferative responses of human lymphocytes was
`examined. The results showed that of the three tetracycline analogues studied, doxycycline
`possessed the most potent inhibiting effects. This occurred at drug concentrations (1-10 /lg/ml)
`easily attainable in serum during conventional dosage schedules. Other investigations have
`shown that tetracyclines also interfere with neutrophil function. Taken together, these findings
`may have clinical significance. Recovery from serious infections generally requires some minimal
`host immune responses, and the immunosuppressive side-effects of tetracyclines may have detri-
`mental effects on patients with debilitating illnesses or impaired immunological defence mecha-
`nisms. Furthermore, tetracyclines may share some common properties of conventional immuno-
`suppressive drugs, such as cytotoxicity, teratogenicity and cancerogenicity. The long-term use of
`tetracyclines for conditions such as chronic bronchitis, bronchiectasis and acne vulgaris needs to be
`re-examined.
`
`INTRODUCTION
`The tetracyclines are a group of antibiotics with broad spectrum antimicrobial activity enjoying wide-
`spread clinical use (Weinstein, 1975). Previous studies have shown that tetracyclines exert inhibitory
`effects on neutrophil function (Munoz & Geister, 1950; Forsgren, Schmeling & Q!tie, 1974; Martin
`eta/., 1973; 1974; Rubinstein & Pelet, 1973; Hill eta/., 1974). The effect of tetracyclines on lymphocyte
`function has not been studied. We have therefore studied the effects of tetracycline analogues on mitogen-
`induced lymphocyte proliferative responses, the in vitro correlates of immune responsiveness.
`
`MATERIALS AND METHODS
`The tetracycline analogues (tetracycline, doxycycline and oxytetracycline} were kindly provided by Pfizer Prop. Ltd., New
`South Wales, Australia. They were dissolved in RPMI 1640 tissue culture medium on the day of each experiment.
`Lymphocytes were purified from heparinized blood of healthy adult donors by Hypaque-Ficoll centrifugation (Boyum,
`1968}. The cells were washed three times and resuspended in RPMI1640 medium containing 10% heat-inactivated foetal
`calf serum. Preliminary studies have established that the tetracycline analogues, at the concentrations used in these experi-
`ments, were not toxic to lymphocytes for up to 4 days in culture, as assessed by trypan blue dye exclusion,
`Lymphocyte transformation studies were performed by a microtechnique described previously (Thong et al., 1973).
`Briefly, sterile microtitre plates were used for cell cultures. Each round-bottom well received 2 x 105 lymphocytes in 0·1 ml
`volume and either 0·05 ml of phytohaemagglutinin (PHA} or pokeweed mitogen (PWM}. To the test wells Wf!re added 0·05
`ml of one of the tetracycline analogues to reach a final concentration of 1·0 pg, 4·0 pg or 10 pg{ml. Control wells received
`0·05 ml of medium only. The final concentrations for PHA (1·0 pgfml} and PWM (SO pgfml) used in these experiments were
`previously determined to produce optimal stimulation.
`
`Correspondence: Dr Y. H. Thong, Department of Paediatrics, The University of Adelaide, The Adelaide Children's
`Hospital, North Adelaide, 5006 South Australia.
`0099-9104{79/003()...{)#3$02.00 @ 1979 Blackwell Scientific Publications
`443
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1030
`
`Exh. 1030
`
`

`
`444
`Y. H. Tltong CS A. Ferrante
`The microtitre plates were incubated for 72 hr at 37°C in a 5% C02-air atmosphere and high humidity. 6 hr prior to
`harvesting, 1·0 pCi of 3H-thymidine was added to each weil. Harvesting was performed with the aid of a Skatron multiple
`sample harvester. The cells were aspirated onto glass-fibre filters, automatically washed with 0·015 M saline and then dried for
`quantification of radioactive uptake in a Packard Tricarb liquid scintillation spectrometer.
`Additional experiments were performed to examine the effect of delayed addition of doxycycline on lymphocyte prolifera·
`tive responses. A separate set of experiments was performed to determine whether the inhibitory effects of doxycycline on
`lymphocyte transformation could be reversed by washing. A further set of experiments was also conducted to assess blasto-
`genic responses by direct microscopy of stained samples, in order to confirm that inhibition of 3H-thymidine uptake correlates
`with suppression of blastogenesis.
`
`RESULTS
`Among the three tetracycline analogues, doxycycline was found to produce the greatest suppression of
`3H-thymidine uptake in both PHA-stimulated (Table 1), and PWM-stimulated cultures (Table 2).
`
`T ABLE 1. Effect of tetracycline analogues on PHA-induced human 1ymphocyte proliferative responses
`
`Tetracycline
`
`Doxycycline
`
`Oxytetracycline
`
`Drug
`concentration
`(pgfml)
`
`Ctfmin 3H-
`thymidine
`uptake
`
`Percentage
`inhibition
`
`Ct{min 3H-
`thymidine
`uptake
`
`Percentage
`inhibition
`
`Ctfmin 3H·
`thymidine
`uptake
`
`Percentage
`inhibition
`
`1
`4
`10
`
`35,990± 10,425
`31,569± 8644*
`32,030± 8864*
`
`15·4
`25·0
`24·0
`
`34,592± 10,541
`26,849± 8299t
`2282±560t
`
`18·7
`35·8
`94·4
`
`35,696± 6705
`30,749± 8977*
`31,945± 8395*
`
`14·4
`26·4
`23·0
`
`Results represent mean± s.d. of nine experiments using cells from nine different donors. Uptake of 3H-thymidine in
`control cultures was 42,288± 8127 ct{min (mean± s.d.).
`* P< 0·01.
`t P< 0·001.
`
`T ABLE 2. Effect of tetracycline analogues on PWM-induced human lymphocyt~ proliferative responses
`
`Tetracycline
`
`Doxycycline
`
`Oxytetracycline
`
`Drug
`concentration
`(pg/ml)
`
`Ctfmin 3H-
`thymidine
`uptake
`
`Percentage
`inhibition
`
`Ct/min 3H-
`thymidine
`uptake
`
`Percentage
`inhibition
`
`1
`4
`10
`
`17,749± 6478
`13,888± 5063*
`12,042±_5349-
`
`4·8
`27·3
`36·2
`
`17,834±7041
`12,378±4193*
`1423± 1071
`
`5·0
`31·0
`92·2
`
`Ctfmin 3H-
`thymidine
`uptake
`
`Percentage
`inhibition
`
`20,975±9468
`17,217±7602
`16,295± 7537
`
`0
`9·1
`13·6
`
`Results represent mean± s.d. of nine experiments using cells from nine different donors. Uptake of 3H-thymidine in
`control cultures was 19,358±9665 ctfmin (mean±s.d.).
`* P< 0·01.
`t P< 0·001.
`
`At a concentration of 10 Jlg/ml, percentage inhibition of 3H-thymidine uptake in PHA-stimulated
`cultures was 94·4 in doxycycline-treated cultures, compared to 24·0 in tetracycline-treated and 23·0 in
`oxytetracycline-treated cultures. In PWM-stimulated cultures percentage inhibition of 3H-thymidine
`uptake was 92·2 in the presence of doxycycline, compared to 36·2 in the presence oftetracycline, and 13·6
`in the presence of oxytetracycline.
`The inhibitory effect of dQxycycline was also dose-dependent (Tables 1 and 2). This inhibitory effect
`was found tobe due to a direct, inhibition ofblast transformation. At 10 Jlgfml concentration, the blasto-
`
`Exh. 1030
`
`

`
`Tetracyclines and lymphocyte Iransformation
`TABLE 3. Effect of immediate and delayed addition of doxycycline on mitogen-
`induced human lymphocyte proliferative responses
`
`445
`
`Time of addition of
`doxycycline (10 Jlg/ml)
`
`PHA
`ct{min 3H-thymidine
`uptake
`
`PWM
`ct{min 3H-thymidine
`uptake
`
`0
`24 hrs
`48 hr
`None added
`
`3102± 198
`3808±575
`5489±683
`36,946± 6601
`
`1100± 170
`492±38
`1508±419
`16,063± 3252
`
`Results represent mean± s.d. of three experiments using three different donors.
`
`genic index in PHA-stimulated cultures was 0· 5 in the presence of doxycycline compared to 26·1 in the
`control; in PWM-stimulated cultures it was 2·1 in the presence of doxycycline compared to 17·0 in the
`control.
`Some characteristics of this inhibitory effect have been defined. In both PHA- and PWM-treated
`cultures, marked suppression of 3H-thymidine uptake occurred even when doxycycline was added 48 hr
`after the start of experiments (Table 3). The inhibitory effect of doxycycline could be reversed completely
`by washing {Table 4) suggestive of a Iack of tight binding to membrane receptors.
`
`TABLE 4. Reversibility of inhibition of mitogen-induced human lymphocyte
`responses by doxycycline
`
`Treatment
`(doxycycline 10 Jlg/ml)
`
`PHA 3H-thymidine
`uptake
`
`PWM 3H-thymidine
`uptake
`
`Unwashed
`Washed
`Untreated control
`
`2158±631
`22,618± 3519
`26,922± 4236
`
`1377±660
`11,042± 1619
`13,633±2754
`
`Results represent mean± s.d. of triplicate samples.
`In these experiments, lymphocytes were incubated in the presence of
`doxycycline for 1 hr, and then washed three times prior to culture with
`mitogens.
`
`DISCUSSION
`The results of the present sturlies indicate that doxycycline, and to a much lesser extent, tetracycline
`and oxytetracycline, exettapotent inhibitory effect on mitogen-induced lymphoproliferative responses of
`human lymphocytes. Other investigators have also reported that tetracyclines adversely affect neutrophil
`chemotactic (Munoz & Geister, 1950; Forsgren et al., 1974), phagocytic and metabolic function (Rubin-
`stein & Pelet, 1973; Hili et al., 1974), as weil as the bactericidal effect of serum (Forsgren & Gnarpe, 1973).
`Taken together, these observations may have clinical relevance. Since recovery from serious infections
`generally requires at least some minimal host immune responses, these immunosuppressive properties
`of tetracyclines may be detrimental to patients with debilitating diseases or impaired immunological
`defence mechanisms.
`The tetracyclines may also share some common properties of conventional immunosuppressive drugs,
`such as cytotoxicity, teratogenicity and cancerogenicity (Meischer, Gerebtzoff & Lambert, 1976). The
`clinical observation of acute hepatic necrosis in patients treated with tetracyclines may be a manifestation
`of cytotoxic side-effects (Schultz et al., 1963; Lloyd-Still, Grand & Vawter, 1974). Tetracyclines traverse
`the placenta readily and teratogenic effects on rats and humans have been documented (Carter & Wilson,
`
`Exh. 1030
`
`

`
`Y. H. Thong (5 A. Perrante
`446
`1962; Cohlan, Beverlander & Tiamsic, 1963). The cancerogenic potential of tetracyclines has not yet been
`realised clinically. These potential hazards of tracyclines are increased, especially in clinical situations of
`chronic bronchitis and acne vulgaris, where tetracyclines are prescribed for long-term usage.
`The mechanism by which tetracyclines adversely affect immune function is not weil understood. This
`may be related to its metabolic effects on microorganisms. In this respect, its principal action appears to
`be the inhibition of protein synthesis (Gale et al., 1972). At higher concentrations (50-100 p,g/ml), how-
`ever, tetracyclinealso inhibits DNA synthesis and alters the membrane properties of Eschericia coli and
`Bacillus subtilis (Pato, 1977). The relevance of this observation to human lymphocytes is not clear, since
`the effects on human lymphocytes were seen with low concentrations of 1-10 p,g/ml. Further studies are
`needed to elucidate the mechanism of immunosuppression by tetracyclines.
`
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