Cytokine production in saquinavir treated mice

Cytokine production in saquinavir treated mice

Int. J. lmmunopharmac., Vol. 19, No. 4, pp. 243-248, 1997 © 1997InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin G...

428KB Sizes 8 Downloads 89 Views

Int. J. lmmunopharmac., Vol. 19, No. 4, pp. 243-248, 1997 © 1997InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin Great Britain 0192-0561/97 $17.00+ .00

Pergamo

PIh S0192-0561 (97)00031-3

CYTOKINE PRODUCTION IN SAQUINAVIR TREATED MICE ROBERTA PACIFICI*, SIMONETTA DI CARLO, ANTONELLA BACOSI, SIMONA PICHINI and PIERGIORGIO ZUCCARO Clinical Biochemistry Department, Istituto Superiore di Sanit/t, V. le Regina Elena 299, 00161 Roma, Italy (Received21 February 1997; revised 11 July 1997)

Abstract--The effects of chronic and acute saquinavir treatment on murine cytokine production were investigated in both plasma and splenocyte cultures. Cytokine plasma levels were below the detection limit in both saquinavir treated mice and in control mice with the exception of IFN-7, whose levels were detectable in 15 day treated mice. Saquinavir was found to increase interferon ~ (1FN-~) and interleukin-2 (IL-2) production from stimulated splenocytes. Saquinavir also caused a marked reduction of transforming growth factor-ill (TGF-fl0 in supernatants of splenocytes cultures. Conversely, interleukin-lfl (IL-lfl), interleukin-10 (IL-10) and tumor necrosis factor-~ (TNF-c0 production was not modified by drug treatment. In the present paper we hypothesized that this protease inhibitor may be involved in the immunoregulatory cytokine network. © 1997 International Society for Immunopharmacology. Keywords: proteinase, inhibitor, cytokine

Drugs that use alternative viral targets have been developed for the treatment of HIV-infected patients due to the limited success of nucleoside reverse transcriptase inhibitors (zidovudine, didanosine, etc.). The proteinase enzyme of HIV is essential for the normal replication cycle of the virus and it is indeed a potential target for antiretroviral therapy (Kramer et al., 1986). Saquinavir (Ro31-8959, Roche) was the first inhibitor of HIV proteinase to enter clinical trials. Proteinase is required for post-translational processing of the Gag and Gag-Pol polyprotein to yield structural proteins of the viral core, as well as essential enzymes including reverse transcriptase and the proteinase itself (Ratner et al., 1985). Inhibition of this enzyme in vitro leads to the generation of immature non-infectious virions and the subsequent interruption of viral spread (Ashorn et al., 1990; McQuade et al., 1990). The proteinase enzyme has been categorized as an aspartic proteinase (Pearl & Taylor, 1987). Although HIV proteinase is not found in uninfected cells, there are several cell-derived aspartate proteinases that serve important homeostatic functions. These include the digestive enzymes pepsin and gastricsin, renin and cathepsins D and E (Kay et al., 1988). Each of these enzymes shares with HIV proteinase the presence of a catalytically essential pair of

aspartyl residues at their active site, hence the designation aspartate proteinase. Because proteolytic activity is essential for several physiological activities of immunocompetent cells, it has been suggested that inhibition of aspartate proteinase might have serious consequences on immune function (Takahashi et al., 1989). Studies looking at HIV replication during infection of different cellular models showed that cytokines mediate the regulation of HIV expression (Farrar et al., 1991). The purpose of the studies described in this report was to determine the eventual interference of saquinavir with cytokine production in the animal model. The tested cytokines were the principal inflammatory cytokines like interleukin-lfl (IL-lfl), interleukin-2 (IL-2), tumor necrosis factor-~ (TNF-~t), and interferon 7 (IFN-7), known to play a crucial role in the pathogenesis of many infections and autoimmune deseases (Schreurs, 1993) and pleiotropic antiinflammatory cytokines like interleukin-10 (IL-10) and transforming growth factor-ill (TGF-fl0, known to produce profound effects on cells involved in the immune response (Whiteside, 1994). Acutely and chronically saquinavir treated mice were used to examine plasma and cell culture supernatants secretion of the above-mentioned cytokines.

*Author to whom correspondence should be addressed. 243

244

R. PACIFICI et al. EXPERIMENTAL P R O C E D U R E S

M/de Male C57BL/6 inbred mice (Charles River, Calco, Como, Italy), 8-10 weeks old, weighing 18-20 g were used. Animals were housed five per cage at 21 +_ I°C room temperature, 50% humidity, 12h/day artificial lighting with access to a Min Rieper pellet diet and water ad libitum. Drug treatments Saquinavir mesylate (Roche, Basel, Switzerland) was dissolved in 0.9% saline solution. Five mice for group were treated orally by gavage with different dosage ratios (5 and 10mg/kg/day). The dose of 10 mg/kg is known to produce in rodents plasma levels that have anti-viral IC90 values (Noel et al., 1992). Drugs were administered daily at 9.30a.m. for 1, 7 and 15 days consecutively. A control group received 0.9% saline solution. Sample collection Blood was collected from the retro-orbital plexus (2.5 ml pooled from five mice) using capillary tubes prefilled with 12 U sodium heparin. Plasma was separated by centrifugation (300rpm for 10min) and stored at - 80°C until analysis. For murine spleen cell preparation, spleens were aseptically removed from sacrificed mice with scissors and forceps in cold phosphate-buffered saline (PBS, JRH Bioscience, Lenexa, KS, U.S.A.) and gently homogenized with a loose Teflon pestle. After allowing the tissue debris to settle for 3-5 min at 4°C, the cells were collected and washed three times with cold PBS. Red blood cells were removed by hypotonic lysis. Splenocytes were adjusted at a final concentration of 1 × 106/well in 96well microtitre plates (Costar, Data Packaning Corp., Cambridge, MA, U.S.A.) in RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum, l-glutamine and penicillin/streptomicin (Gibco Laboratories, Grand Island, NY, U.S.A.). Splenocytes were collected 18 h after the last treatment. A first set of splenocytes were stimulated at 37°C in 5% CO: incubator 36 h with phytohaemagglutinin (PHA, 1/tg/ml, Sigma Chemical Co., St Louis, MO, U.S.A.) for the induction of IL-2 and IL-10 and 72 h for the induction of IFN ~. A second group of splenocytes were stimulated 24h by lypopolysaccharide from Escherichia coli (LPS, 10/~g/ml, Sigma Chemical Co.) for the induction for IL-lfl, TNF7 and TGF-fi, production. A third set of splenocytes were cultured simultaneously in triplicate without stimulation to deter-

mine the spontaneous production of cytokines. After incubation, the culture supernatants were collected and stored at - 80°C for the quantitation ofcytokines. Cytokine assay For quantitative measurement of murine IL-1 fl, I L2, IL-10, TNFa, IFNT, and biologically active TGF-fi~ in plasma and in supernatants of splenocytes cultures, specific solid-phase ELISA assays, employing the multiple antibody sandwich principle, were used (Genzyme Inc., Cambridge, MA, U.S.A.). The tests were performed according to the supplier's instructions with the exception of TGF-fi~ assay, which is sold for human tissues and was adapted in our laboratory for murine tissues testing cross-reactivity, specificity, and sensitivity parameters. Samples from control and treated mice and interleukin standards were assayed simultaneously, in triplicate, in 96-well microtitre plate pre-coated with monoclonal anti-ILs. The standard curve for IL-lfl assay ranged between 0.01 and 0.4 ng/ml; that for IL2 assay between 0.01 and 1 ng/ml; that for IL-10 assay between 0,03 and 0.2ng/ml; that for TNF~ assay between 0.03 and 0.6 pg/ml; that for IFN7 assay 0.02 and 1.6ng/ml; and that for TGF-fll assay 0.1 and 0.6 ng/ml. Tissue culture supernatants were diluted in wash buffer and the dilution factor was considered for the calculation of IL amount. The sensitivity of assay was 0.01 ng/ml for IL-lfl, IL-2, IL-10 and TNF~, 0.005 ng/ml for 1FNy and 0.05 ng/ml for TGF-fl,. The specificity of monoclonal anti-mouse-IL was tested in our laboratory. Negative reactions with other murine cytokines such as IL-la, IL-4, IL-6, and lymphocyte inhibitory factor were found. Assay performance was tested using three concentrations of cytokines in plasma and in culture medium throughout the procedure. Mean intra- and inter-assay coefficients of variation were always less than 5%. Statistical analysbs Values are presented as mean _+ S.D. All means were calculated from determinations obtained in three different experiments. The impaired Student t-test was used to compare the results obtained in treated mice with those of control group.

RESULTS The effect of acute (1 day) and chronic (7 and 15 day) saquinavir treatment was investigated by measuring IL-1/~, IL-2, IL-10, TNF~, IFNy, and TGF-fi~ levels in plasma and cultured spleen cells from mice.

Cytokine Production in Saquinavir Treated Mice In all the experimental conditions, cytokines plasma levels were below the detection limit in both saquinavir treated mice and control mice with the exception of I F N - 7 levels in 15 day treated mice that resulted to be 0.25 and 0.40ng/ml for 5 and 10mk/kg treatment, respectively. Acute and chronic treatment with saquinavir at 5 and 10 mg/kg doses did not significantly alter IL-1 fl, IL-10 and TNF~ production in murine cells stimulated with PHA or LPS in vitro (data not shown). Conversely, IL-2 production in mitogen-stimulated splenocytes significantly increased by 15 day treatment with saquinavir (Fig. 1). The increase (125 and 117% for 5 and 10 mg/kg, respectively) was not dosedependent. Likewise, the secretion of IFN-7 was significantly higher in cultured splenocytes from saquinavir treated mice as compared to the controls. In particular, IFN7 secretion by PHA-stimulated splenocytes increased to 61% of the control level after a 7 days treatment with 10mg/kg saquinavir, whereas 5mg/kg dosage produced a lesser increase. In the case of 15 day treatment, both 5mg/kg and 10mg/kg dosage similarly increased IFN-7 secretion in splenocytes of treated mice (Fig. 2). TGF-flj release by C57BL/6 mice splenocytes activated by LPS did not change significantly after a 7 day treatment with both 5 and 10mg/kg saquinavir. Conversely, after 15 days of treatment, TGF-fll release was significantly inhibited in splenocytes of mice tre-

_

245

ated with 10 mg/kg saquinavir, while it was not altered by the 5 mg/kg treatment (Fig. 3).

DISCUSSION For an HIV proteinase inhibitor, nonspecific inhibition of cellular aspartate proteinases (pepsin, gastricsin, renin, and especially cathepsins D and E), might have the potential to result in adverse effects (Kay et al., 1988). For example, IL-lfl is synthesized as a 31 kDa precursor (pre-IL-lfl) and is believed to be susceptible to in vitro cleavage by several proteases (Kostura et al., 1989; Black et al., 1989). However, we observed that saquinavir did not affect the secretion of IL-lfl in the culture medium. This result was also in agreement with the observations of Bugelski et al. These authors demonstrated that another inhibitor of proteinase, SKaF107461, was without effect on 17 kDa IL-lfl secretion (Bugelski et al., 1992). These results were not unexpected because saquinavir is known to be a selective inhibitor, and indeed endopeptidase cleavage at the amino terminal of proline is a very rare event in mammalian biochemistry, implying that interference at this site will give selective inhibition of viral rather than mammalian biochemistry, implying that interference at this site will give selective inhibition of viral rather than mammalian proteinase (Craig et al., 1991).

o Control • Saquinavir 10 mg/kg/da Saquinavir 5 mg/kg/day

0,5-

TT

J

!

0,1I

I

I

1

7

15

Days

Fig. 1. Time and dose-related effect of saquinavir treatment on in vitro IL-2 production. Splenocytes obtained from mice treated with saquinavir 10 mg/kg/day (.), 5 mg/kg/day (A), and from control mice (o) were stimulated in vitro with PHA. Five mice per experimental point were used, the cells were pooled before seeding in culture. Three different experiments were performed. Results are expressed as mean _+S.D. ***p < 0.001; n.s. not significant.

246

R. PACIFICI et al.

25-

o Control • Saquinavir 10 mg/kg/day Saquinavir 5 mg/kg/day

***

20~15-

n

~

~

~10-

1'5

Days

Fig. 2. Time and dose-related effect of saquinavir treatment on in vitro IFN-y production. Splenocytes obtained from mice treated with saquinavir 10mg/kg/day ('), 5 mg/kg/day (A), and from control mice (o) were stimulated in vitro with PHA. Five mice per experimental point were used, the cells were pooled before seeding in culture. Three different experiments were performed. Results are expressed as mean _+S.D. **p < 0.05, ***p < 0.001; n.s. not significant.

7

n,s. ~l?.s•

n,s•

n s

I

_.2

s

6-

,~'~42-

o Control • Saquinavir 10 mg/kg/day A Saquinavir 5 mg/kg/day

Days Fig. 3. Time and dose-related effect of saquinavir treatment on in vitro TGF-[3] production. Splenocytes obtained from mice treated with saquinavir 10mg/kg/day ('), 5 mg/kg/day (A), and from control mice (o) were stimulated in vitro with LPS. Five mice per experimental point were used, the cells were pooled before seeding in culture. Three different experiments were performed. Results are expressed as mean _+S.D. ***p < 0.001; n.s. not significant. In o u r experimental conditions, chronic saquinavir t r e a t m e n t caused a significant e n h a n c e m e n t of I F N - 7 in stimulate cell culture a n d in serum. It is interesting to note t h a t IFN-~, is a n antiviral i m m u n e m o d u l a t o r y protein t h a t regulates the M H C class II antigen prese n t a t i o n (Kadereit et al., 1993). The antigen-pre-

senting cells ( A P C ) c o m p a r t m e n t ( s ) t h r o u g h which antigens are re-expressed on the surface, in association with m a j o r histocompatibility complex ( M H C ) class II antigens, have been s h o w n to c o n t a i n proteases like cathepsins B, D a n d L (Guagliardi et al., 1990; H a r d i n g et al., 1990).

Cytokine Production in Saquinavir Treated Mice The exact nature of the proteases involved in generating the epitopes recognized by T cells depends on the particular protein being processed and on the genetic make-up of the individual (MHC class II) (Takahashi et al., 1989). A recent study showed that exposure to IFN-7 causes the selective enhancement of the expression and activities ofcathepsins in human and mouse monocytes lines (Lah et al., 1995). Our result showed also that IL-2 release is increased in stimulated cell culture after 15 days of saquinavir treatment. This delayed increase of IL-2 could be the indirect response of the earlier and massive IFN- 7 release. Indeed, IFN-7 is the only cytokine which appears in detectable amounts in serum. Conversely, 15 days of saquinavir treatment caused a significant decrease of TGF-/~ release in stimulated cell culture. Actually, there is an increase in type 1 cytokines IFN- 7 and IL-2 and a decrease in TGF-/~ production, a type 2 cytokine that is known to suppress type 1 cytokines, by mitogen stimulated splenocytes at day 15 post treatment, while this correlation is not observed at day 10. IFN-7 is known to play a critical role both as an inducer of the differentation of C D 4 + T cells to the Th 1 phenotype characterized by the production of IL2 and as the major product of Thl cells (Young & Hardy, 1995). Since the importance of the ratio of Thl to Th2 lymphocytes in influencing the course of an infectious disease has been reported (Del Prete, 1992), the stimulation of IFN- 7 by saquinavir treatment could be particularly useful in precocious treatment of HIV seropositive patients if these experimental observations can be confirmed in humans. Furthermore certain cytokines, particularly interferons, have been found to down-regulate HIV expression in some situations.

247

On the other hand, it is intriguing to hypothesize a theoretical link between a decrease in TGF-fl following saquinavir treatment and the fact that purified human monocytes infected in vitro with HIV-1 secreted TGF-fl (Wahl et al., 1991) and that elevated levels of TGF-fl could be detected in serum or plasma of patients with AIDS (Allen et al., 1991). Moreover, protease and inhibitor of proteases are controlled by certain cytokines, chemokines and growth factors and all in concert play critical parts in cellular interactions. TGF-/~ stimulate the synthesis of extracellular matrix (ECM) components reducing the synthesis of proteases that breakdown ECM components and increasing the synthesis of inhibitors of proteases (Lawrence, 1995). Although many questions remain unanswered, changes in spleen cells composition of saquinavir treated mice could explain a modified production ofcytokines. Indeed, further studies are required to elucidate this point even though analysis of subclasses of lymphocytes in the murine spleens following 7 and 15 days of drug treatment did not show a significant variation in the absolute number of L3T4 (helper T cells) and LyT2 (suppressor/cytotoxic T cells) positive cells when compared to control mice (data not shown). Finally, given the ubiquitous nature of these soluble mediators of immune response and their multiple biological activities, it could be of interest to investigate the expression of activation markers on CD4 ÷ and CD8 + cells that might help to explain synthesis levels of cytokines. In future clinical trials, the monitoring of cytokine production might be important in HIV positive patients to obtain a more true picture about cytokine assessment and saquinavir treatment efficacy.

REFERENCES

Allen, J. B., Wong, H. 1., Guyre, P. M., Simon, G. 1. & Walh, S. M. (1991) Association of circulating receptor FcyRIIIpositive monocytes in AIDS patients with elevated levelsof transforming grow factor-/L J. Clin. Invest., 87, 1773 1779. Ashorn, P., McQuade, T. J., Thaisrivongs, S., Tomasselli, A. G., Tarpley, W. G. & Moss, B. (1990) An inhibitor of the protease blocks maturation of human and simian immunodeficiencyviruses and spread of infection. Proc. Natl. Acad. Sci. USA, 87, 7472-7476. Black, R. A., Kronbelm, S. R., Merriam, J. G., March, C. J. & Hopp, T. P. (1989) A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-lfl. J. Biol. Chem., 264, 5323-5326. Bugelski, P. J., Kaplan, J. M., Hart, T. K., Miller, J., Laydon, J. T., Lee, J. C., Dreye, G. B. & Kirsh, R. (1992) Effect of a human immunodeficiency virus protease inhibitor on human monocyte function. A I D S Res. Hum. Retroviruses, 8, 1951-1958. Craig, J. C., Duncan, I. B., Hockley, D., Greif, C., Roberts, N. A. & Mills, J. S. (1991) Antiviral properties of RO 31-8959, an inhibitor of human immunodeficiencyvirus (HIV) proteinase. Antiviral Res., 16, 295-305. Del Prete, G. D. (1992) Human Thl and Th2 lymphocytes: their role in the pathophysiology of Actopy. Allergy, 47, 450455.

248

R. PACIFICI et al.

Farrar, W. L., Korner, M. & Clouse, K. A. (1991) Cytokine relugation of human immunodeficiency virus expression. Cytokine, 3, 531 542. Guagliardi, L. E., Koppelman, B., Blum, J. S., Marks, M. S., Creewell, P. & Brodsky, F. M. (1990) Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment. Nature, 343, 133 139. Harding, C. V., Unanue, E. R., Slot, J. W., Schwartz, A. L. & Geuze, H. L. (1990) Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II peptide complexes during antigen processing. Proc. Natl. Aead. Sei. USA, 87, 553-557. Kadereit, S., Gewert, D. R., Galabru, J., Hovanessian, A. G. & Meurs, E. F. (1993) Molecular cloning of two new interferoninduced, highly related nuclear phosphoproteins. J. Biol. Chem., 268, 24432-24441. Kay, J., Jupp, R. A., Norey, C. G., Richards, A. D., Reid, W. A., Taggart, R. T., Samloff, I. M. & Dunn, B. M. (1988) Aspartate proteinases and inhibitors for their control in health and disease. Adv. Exp. Med. Biol., 240, 1-11. Kostura, M. J., Tocci, M. J., Limjuco, G., Chin, J., Cameron, P., Hillman, A. G., Chartrain, N. A. & Schmidt, J. A. (1989) Identification of a monocyte specific pre-interleukin-1/~ convertase activity. Proc. Natl. Acad. Sci. USA, 86, 5227-5231. Kramer, R. A., Schaber, M. D., Skalka, A. M., Gangnly, K., Wong-Staal, F. & Reddy, E. P. (1986) HTLV lII in Gag protein is processed in yeast cells by the virus pol-protease. Science, 231, 158(~1584. Lah, T. T., Hawley, M., Rock, K. L. & Goldberg, A. L. (1995) Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages. FEBS Lett., 363, 85-89. Lawrence, D. A. (1995) Transforming growth factor/~: an overview. Kidney Int., 9, s19-s23. McQuade, T. J., Tomasselli, A. G., Liu, L., Karacostas, V., Moss, B., Sawyer, T. K., Heinrikson, R. L. & Tarpley, W. G. (1990) A synthetic HIV protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science, 247, 454-456. Noel, A. R., Craig, J. C. & Duncan, 1. B. (1992) Novel approaches in HIV chemoterapy. Biochem. Soc. Trans., 20, 513 516. Pearl, L. H. & Taylor, W. R. (1987) A structural model for HIV proteases. Nature, 329, 351 354. Ratner, L., Haseltine, W., Patarca, R., Livak, J. K., Starcich, B., Josephs, F. J., Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Pearson, L. M., Lautenberger, J. A., Papas, T. S., Ghayeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus. Nature, 313, 277-282. Schreurs, J. (1993) Cytokines in infections desease: the battle between host and pathogen. Curr. Opinion Biotechnol., 4, 727 733. Takahashi, H., Cease, K. B. & Berzofsky, J. A. (1989) Identification of proteases that process distinct epitopes on the same protein. J. Immunol., 142, 2221 2229. Wahl, S. M., Allen, J. B., McCartney-Francis, N., Morganti-Kossman, M. C., Ellingsworth, L., May, U. E. H., Mergenhagen, S. E. & Orenstein, J. M. (1991) Macrophage- and astrocyte-derived transforming growth factor/~ as a mediator of central nervous system dysfuction in acquired immune deficiency syndrome. J. Exp. Med., 173, 981-991. Whiteside, T. L. (I 994) Cytokine measurements and interpretation of cytokine assays in human desease. J. Clin. Immunol., 14, 327-339. Young, H. A. & Hardy, K. J. (1995) Role of interferon-v in immune cell regulation. J. Leukoc. Biol., 58, 373-381.