key: cord-007646-yh8zi1ee
authors: Weiss, R.C.; Oostrom-Ram, T.
title: Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats()
date: 2002-11-12
journal: Vet Immunol Immunopathol
DOI: 10.1016/0165-2427(90)90017-m
sha: 
doc_id: 7646
cord_uid: yh8zi1ee

The effect of recombinant human interferon-alpha (rHuIFN-α) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 10(4) or 10(3) International Units (U) of rHuIFN-α for 24 h significantly suppressed (P<0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10–10(−3) U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 10(4)–10(2) U (P<0.001) or 10 U (P<0.05) of rHuIFN-α/ml resulted in a significant decrease in incorporation of [methyl-(3)H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-α doses of 10(4) U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects. To investigate the dose-response effects of rHuIFN-α in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) U/kg), moderate dose (10(4) U/kg), or a relatively low dose (10(2) U/kg) of rHuIFN-α. Cats inoculated with 10(6) U ofrHuIFN-α/kg had significantly reduced (P=0.037) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 10(4) or 10(2) U of rHuIFN-α/kg significantly enhanced (P=0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-α given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing.

gen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 104 or 103 International Units (U) of rHuIFN-o~ for 24 h significantly suppressed (P < 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10-10 -3 U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 104-102 U (P < 0.001 ) or 10 U (P< 0.05) of rHuIFN-o~/ml resulted in a significant decrease in incorporation of [methyl-3H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-o~ doses of 104 U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects.

To investigate the dose-response effects of rHuIFN-~ in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose ( 106 U/kg), moderate dose ( 104 U/kg), or a relatively low dose ( l0 s U/kg) of rHuIFN-ol. Cats inoculated with 108 U of rHuIFN-~fkg had significantly reduced (P = 0.037 ) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 104 or 102 U of rHuIFN-o~/kg significantly enhanced (P--0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-o! given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing. INTRODUCTION Interferons (IFN) are a group of cellular proteins that inhibit viral replication, modulate immune responses, inhibit normal cellular division and have antitumor activity (Borden and Ball, 1981 ) . There are several classes of IFN ( a, fl and y) which differ on the basis of their antigenic, biological and physic ochemical properties (Mannering and Deloria, 1986) . Because IFN (particularly human IFN-c~ ) has antiviral and immunomodulatory activities that cross species lines (Pallikoff et al., 1962; Desmyter et al., 1968; Gresser et al., 1974; Krakowka et al., 1988) and because recombinant DNA-produced human IFN is now available in relatively large quantity, the use of human IFN in veterinary medicine has increased. Human IFN-c~ has been used clinically in cattle afflicted with respiratory diseases (Roney et al., 1985; Cummins and Hutcheson, 1986) , in cats infected with either feline leukemia virus (FeLV) or feline infectious peritonitis virus (FIPV) , and in dogs infected with canine parvovirus (Dr. J. Cummins, unpublished data, 1986) . Dosages of IFN used clinically in domestic animals have been empirical and extrapolated largely from studies in persons or mice using interferon preparations that can differ in purity and potency. Unfortunately, studies of the immunological effects of homologous or heterologous IFN in domestic animals have been lacking; the effects of human IFN-c~ in vitro on canine immune responses, however, have recently been reported (Krakowka et al., 1988) .

Our interest in studying IFN is related to its application clinically as an antiviral and immunomodulating drug in cats with viral diseases. The effects of heterologous IFNs on feline immune responses in general are not known. Resistance to diseases such as FIP is associated with effective cell-mediated immunity (CMI) (Pedersen and Floyd, 1985; ; cats infected with other viruses like FeLV may have severely impaired T-cell responses (Rojko and Olsen, 1984 ) . Obviously, an understanding of the relationship between IFN dosage and modulation of CMI is necessary prior to recommending IFN as treatment for viral diseases where stimulation of CMI is required. Mitogen-induced lymphocyte blastogenesis, which is a widely used in vitro assay for evaluation of CMI (Oppenheim and Schechter, 1976) , has been used previously in cats to assess alterations in CMI induced by immunomodulating agents such as cyclosporin (Gregory et al., 1987 ) . In the studies reported here, the effects of doses of human IFN-c~ in vitro and in vivo on lymphocyte blastogenesis in normal cats was investigated.

The cats used in these studies were healthy, 10-12-month-old specific-path-ogen-free (SPF) males and females, weighing approximately 3.5 kg. The cats were purchased from a commercial breeder (Liberty Laboratories, Liberty Corners, NJ ) and were FeLV test-negative (by ELISA) and feline coronavirus antibody-negative prior to the studies. The cats were housed separately in cages located in the Scott-Ritchey Animal Isolation Facility and were tested and cared for according to humane standards as set forth in the "Guide for the Care and Use of Laboratory Animals" (Publication No. 85-23, National Institutes of Health, Bethesda, MD). All experimental protocols were approved by an independent animal welfare committee prior to the studies.

The recombinant DNA-derived (Bgl-II restriction endonuclease-specified) human leukocyte (alpha) hybrid {subtypes A/D) IFN (rHuIFN-a), lot no. RO-23-1740, was kindly supplied by Dr. Richard Cordts, Hoffman LaRoche, Nutley, NJ. The IFN was stored lyophilized (at a concentration of 50X 106 International Units (U)/vial) at -80°C prior to use. The IFN was diluted before use in Hank's balanced salt solution (HBSS; pH 7.2) and was sterilefiltered through a 0.2 ]~M cellulose acetate membrane (Nalge Co, Rochester, NY ). The biological activity of the rHuIFN-c~ (expressed in U/ml of antiviral activity) was determined in the manufacturer's laboratory, using an international reference standard for human leukocyte IFN.

Cats were lightly anesthetized with an intramuscular injection of ketamine hydrochloride (Vetalar; Parke, Davis & Co, Detroit, MI) and blood collected by jugular venipuncture into glass syringes containing heparin (10 units/ml of blood). Peripheral blood lymphocytes (PBL) were isolated and prepared as previously described (Cockerell et al., 1975; Tham et al., 1982) . Briefly, PBL were separated by Ficoll-diatrizoate (Histopaque-1077; Sigma Chemical Co, St Louis, MO) gradient separation and the interface mononuclear cells collected and washed in HBSS. The washed cells were resuspended to 2 X 106 viable cells/ml in tissue culture growth medium (GM) consisting of RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 100 U of penicillin/ml and 100/lg of streptomycin/ml along with 1.0 mM L-glutamine and 25 mM HEPES.

For the in vitro IFN studies, PBL (2.0 X 105 cells, 0.1 ml) were pipetted into wells of sterile 96-well flat-bottom microtitration plates (Corning Glass Works, Corning, NY) and then incubated with GM containing varying amounts of rHuIFN-a or GM only (0.1 ml/well) for 24 h at 37°C in humidified air containing 5% CO2. For the in vivo studies, preincubation of PBL with IFN was omitted. The cell suspensions were then cultured 72 h with either concanavalin A (Con A) (0.6 ttg/well) or pokeweed mitogen (PWM) (0.5 ttg/well), or they were not treated with mitogens (6 replicates/treatment). Previous titrations in feline PBL indicated that the concentrations of mitogen used were optimal for our assay. To each well, 0.5/~Ci (0.025 ml ) of [methyl-3H ]thymidine (specific activity 6.7 Ci/mmol) (Dupont, NEN Research Products, Boston, MA) was added for the last 20 h of incubation. The cultures were then stored at -80 ° C until harvested. Cells were harvested using a semi-automatic multiple cell microharvester (Bellco Glass, Vineland, NJ) so that the cellular proteins were collected onto glass fiber filter paper strips (Bellco). The paper strips were dried at 60 °C for 30 min and the filter discs transferred to scintillation vials into which 5 ml of toluene base cocktail (Scinti Verse II; Fischer Scientific, Fairlawn, NJ) was added. The incorporation of [methyl-3H]thymidine into newly synthesized cellular DNA was quantitated in a liquid scintillation spectrometer (LKB-Wallac Oy, Turku, Finland) using channels ratio method of quench correction. The net counts per minute (c.p.m.) of a total of 6 wells for each variable were averaged to obtain the mean c.p.m. The stimulation index (s.i.) was determined by dividing the mean c.p.m, of the mitogen-stimulated (or rHuIFN-~-stimulated only) cells by the mean c.p.m, of unstimulated (media control) cells. Stock solutions of IFN, thymidine and fetal calf serum were prepared from the same lots, respectively, prior to the studies and only fresh reagents were used.

To determine the potential cytotoxic effect of rHuIFN-c~ on feline PBL, lymphocytes were isolated from eight normal cats as described before and were exposed to varying amounts of rHuIFN-c~ continuously for several days. Briefly, freshly isolated PBL (2.0 × 105 cells, 0.1 ml) were cultured with GM (0.1 ml/ well) containing rHuIFN-c~ (ranges, 104-1.0 U/ml) or GM only for 96 h at 37°C in 96-well microtitration plates (6 replicates/dose). The nonadherent cells were aspirated from a total of 6 wells for each IFN dose; the number of viable cells, determined by trypan blue dye exclusion, were counted in duplicate using a Neubauer hemocytometer. Results were expressed as mean viable cell density (number of viable cells/ml) in rHuIFN-c~-treated or medium control cultures.

A study was designed to evaluate the effects of giving varying amounts of rHuIFN-c~ parenterally on lymphocyte blastogenesis in cats. Cats were randomly assigned to one of three experimental groups and were treated as follows: a high-dose IFN group (n= 10 cats) received a single injection i.m. of rHuIFN-~ at a dosage of 106 U/kg; a moderate-dose IFN group (n=4 cats) likewise received 104 U of rHuIFN-c~/kg; and a low-dose IFN group (n=4 cats) similarly received 102 U of rHuIFN-c~/kg. Three additional cats were inoculated similarly with HBSS (diluent controls). Cats were evaluated by lymphocyte blastogenesis in response to phytomitogens at 24 h postinoculation. In order to minimize normal diurnal variation in test results, all cats were evaluated by lymphocyte blastogenesis at 24-h intervals on 3 successive days preceding inoculation and then 24 h postinoculation. For each group of cats, a single preinoculation mean Con A or PWM response (3-day average) was determined and then compared statistically against the corresponding postinoculation group mean. Each group of cats was evaluated separately on alternate weeks.

The one-tailed Student's t test was used to determine statistical significance between groups. All data analysis was performed using a computerized statistical analysis program (Abstat; Anderson-Bell, Canon City, CO). A conservative number of degrees of freedom was used (i.e., one for each animal rather than one for each well). P values of ~< 0.05 were considered significant.

Incubation of normal feline PBL with doses of rHuIFN-a in vitro suppressed lymphocyte proliferative responses to Con A and PWM (Table 1) . Suppression of blastogenesis was dose-dependent; significant inhibition occurred at relatively high in vitro doses of rHuIFN-c~ (104 or 103 U/ml). Doses of 102 U of rHuIFN-c~/ml also decreased mean Con A or PWM responses, but this effect was not statistically significant. Lower doses of rHuIFN-a (10.0-10-3 U/ml) neither suppressed nor enhanced mitogenesis.

Incorporation of [methyl-3H ] thymidine into newly synthesized cellular DNA in the absence of phytomitogens was significantly inhibited in feline PBL cultures exposed to rHuIFN-~ doses of 104-10.0 U/ml compared to untreated PBL (Table 2) . Mean thymidine incorporation (c.p.m.) in cultures treated with 104-102 or with 10.0 U/ml was approximately 14% or 50%, respectively, of the thymidine incorporation measured in untreated cells.

Varying amounts of rHuIFN-c~ (104-1.0 U/ml) were added to PBL cultures to determine whether direct cytotoxic effects from the IFN itself may have contributed to the dose-related suppression of blastogenesis observed in vitro. Differences in mean viable cell density between untreated PBL cultures and those treated with IFN, however, were not observed. (Fig. 1) . 

To determine the effects of varying amounts of rHuIFN-a in vivo on mitogen-induced blastogenesis, cats in each of three groups were given a single injection of either a high (106 U/kg), moderate (104 U/kg), or relatively low dose (102 U/kg) of rHuIFN-~ and then evaluated 24 h later. To minimize normal diurnal variation in responses, a 3-day average of daily mean responses was determined preinoculation for each group. Day-to-day variation in blastogenic responses of individual cats evaluated on 3 consecutive days of the same week was not significant (P > 0.05, n---18; data not shown). Cats inoculated with a single high dose of rHuIFN-~ had a significant suppression (P= 0.037) in Con A-induced lymphocyte blastogenesis 24 h postinoculation compared to preinoculation values ( Fig. 2A) . Mean PWM responses were also decreased, but this effect was not statistically significant. In contrast to the cats inoculated with a high dose of rHuIFN-~, cats given 104 or 102 U of rHuIFN-c~/kg had significantly enhanced (P=0.008 or 0.05, respectively) blastogenic responses to Con A; PWM responses, however, were unaffected ( Fig. 2B and C ) . Inoculation of cats with HBSS alone had no significant effect on mitogen-induced blastogenesis 24 h postinoculation (data not shown).

The effects of IFN in vitro or in vivo on the immune system are manyfold and sometimes apparently contradictory. Overall, it appears that IFN can either stimulate or suppress various arms of the immune response, depending on the timing of administration and dosage (Epstein, 1977) . IFN in general seems to inhibit immunologic responses when given prior to an immunogen but enhances responses if given some days later (White and Fenner, 1986) . Moreover, high doses of IFN can produce opposite effects or annul the responses obtained with much lower doses. For example, very low doses of IFN-c~ in vitro or in vivo may stimulate development of antibody-forming spleen cells in mice, whereas high doses are immunosuppressive (Braun and Levy, 1972; Epstein, 1977) . The addition of low doses of IFN (10 or 100 units/ml) in the primary mixed lymphocyte reaction increases the cytotoxic response in mice severalfold, but larger doses (104 units/ml ) depress the response (Fradelizi and Gresser, 1982 ) . Similarly, low doses of IFN can enhance lymphocyte blastogenesis in mice or persons, whereas large doses are suppressive (Miorner et al., 1978; Taylor-Papadimitriou, 1980) . Depending on the timing of administration, extremely low doses of IFN-~, fl (2 × 10-lO U) in mice significantly increase the number of antibody-secreting cells in spleen and strongly stimulate cytotoxic activities of allospecific T-cells (Daurat et al., 1988) .

The results-of this study showed that very high doses of rHuIFN-~ either in vitro or in vivo suppressed mitogen-induced lymphocyte proliferative responses in cats. The rHuIFN-~ also directly inhibited the in vitro incorporation of [methyl-3H ]thymidine into newly synthesized cellular DNA of unstimulated PBL and at much lower IFN concentrations than those required to suppress mitogen-induced blastogenesis. The inhibitory effects of various types of IFN on DNA synthesis and lymphocyte blastogenesis after mitogenic stimulation have been described previously in different species, including mice, cattle, and persons (Lindahl-Magnusson et al., 1972; Blomgren et al., 1974; Bielefeldt-Ohman and Babiuk, 1986; Kim et al., 1988; Roth and Frank, 1989 ). IFN has also been shown to directly inhibit thymidine incorporation into the DNA of normal cells, including both lymphoid and epithelial cells, in the absence of mitogenic stimulation (Brouty-Boye and Tovey, 1978; Stadler et al., 1986; Roth and Frank, 1989) . Although the suppressive mechanism (s) associated with rHuIFN-c~ on DNA synthesis and lymphocyte blastogenesis were not investigated in our study, it is possible that high doses of IFN in vitro enhanced lectin-binding on lymphocytes and stimulated suppressor cell activities. IFN can markedly enhance the binding of lectins to lymphocyte membranes (Miorner et al., 1978) , and suppressor cells are activated by high doses of mitogen, particularly Con A (Piguet et al., 1976) .

The IFN-mediated suppression of lectin-induced blastogenesis observed when feline PBL were preincubated 24 h with relatively high doses of rHuIFNa was also seen 24 h after cats were inoculated i.m. with very high doses (106 U/kg) of rHuIFN-a. Significant decreases in blastogenic responses to T-cell mitogens (Con A) in particular were observed after parenteral administration of high-dose rHuIFN-a. Although responses to PWM (which acts both as a B-and T-cell mitogen) (Heegaard and Muller, 1988) were diminished, this effect was not statistically significant, suggesting that T-cell responses perhaps were somewhat more sensitive to the suppressive actions of high doses of IFN in vivo. Curiously, lower parenteral doses of rHuIFN-a (104-102 U/kg) significantly enhanced the blastogenic responses of PBL after stimulation with T-cell mitogens (this phenomenon, however, was not observed when low doses of IFN were incubated in vitro with PBL). An inverse relationship between IFN dose and lymphocyte proliferative responses similar to that which we observed in cats has been documented previously in vitro and in vivo in mice and persons (Miorner et al., 1978; Taylor-Papadimitriou, 1980; Kim, 1988) . Seemingly, a mechanism associated with this phenomenon may have been IFNinduced activation of suppressor cells. Bielefeldt-Ohmann and Babiuk (1986) reported that in vitro treatment of bovine PBL with recombinant bovine IFNgamma (rBoIFN-7) induced suppressor cells which may have competed with interleukin (IL)-2; moreover, the suppression in lymphocyte blastogenesis after in vivo administration of high doses of rBoIFN-7 was found to be reversible by addition of human IL-2 to the lymphocyte cultures. Lower amounts of IFN, however, may actually enhance the cellular immune response by selectively blocking suppressor pathways (Knop et al., 1984) . In support of this theory, Daurat et al. (1988) demonstrated enhanced cytotoxic activities of allospecific T-cells and enhanced cytotoxic responses of NK cells in mice inoculated several times with IFN-a, fl at dosages as low as 2.0 or 2 × 10-lo U. The inverse dose-response effects of IFN, particularly its biologic activity at very low pharmacologic doses, has suggested a predominantly hormone-like action of IFN as a homeostatic regulator of immune functions (Daurat et al., 1988) .

Undoubtedly, an understanding of the dose-response effects of IFN on normal feline immune responses is imperative when considering IFN therapy in cats with disease. Additional in vitro and in vivo studies on the effects of IFN on T-and also B-cell responses in cats will be required so that specific recommendations concerning the use of IFN in feline viral or other diseases rationally can be established.

Alteration of some leukocyte functions following in vivo and in vitro exposure to recombinant bovine alpha-and gamma-interferon

Effect of human leukocyte interferon on the response of lymphocytes to mitogenic stimuli in vitro

Interferon: Biochemical, cell growth inhibitory, and immunologic effects

Interferon preparations as modifiers of the immune response

Inhibition by interferon of thymidine uptake in chemostat cultures of L1210 cells

Phytomitogen-and antigeninduced blast transformation of feline lymphocytes

Low dosage of interferon to enhance vaccine efficiency in feedlot calves

Oral use of human alpha interferon in cats

Immunomodulatory activity of low doses of interferon o~, fl in mice

Human interferon that crosses species lines

The effects of interferons on the immune response in vitro and in vivo

Interferon inhibits the generation of allospecific suppressor Tlymphocytes

Response to isoantigens and mitogens in the cat: Effects of cyclosporin A

Pronounced antiviral activity of human interferon on bovine and porcine cells

Lectins and the immune system

Interferon inhibition of IL-2-mediated lymphocyte proliferation

Inhibition of the T-suppressor circuit of delayed-type hypersensitivity by interferon

The effects of human interferon-alpha upon in vitro canine immune responses

Interferon inhibits DNA synthesis induced in mouse lymphocyte suspensions by phytohaemagglutinin or by allogeneic cells

The pharmacology and toxicology of the interferons: An overview

Regulation of mitogen-induced lymphocyte DNA synthesis by human interferon of different origins

Manual of Clinical Immunology

Tissue specificity of interferons prepared in various tissue cultures

Experimental studies with three new strains of feline infectious virus: FIPV-UCD2, FIPV-UCD3, and FIPV-UCD4

Induction or suppression of B cell proliferation and differentiation by phytohemagglutinin or concanavalin A in mouse spleen cell cultures

The immunobiology of the feline leukemia virus

Effect of human leukocyte interferon on prevention of infectious bovine rhinotracheitis virus infection

Recombinant bovine interferon-y as an immunomodulator in dexamethasone-treated and non-treated cattle

Effect of recombinant alpha A-interferon on DNA synthesis and differentiation of human keratinocytes in vitro

Effects of interferons on cell growth and function

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

Evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity

Effect of interferon or Propionibacterium acnes on the course of experimentally induced feline infectious peritonitis

Medical Virology (3rd Edn

Research program. The authors thank Ms. Lewanne French and Dr. Mitzi Martinez for their assistance.