Journal of Clinical Laboratory Analysis 6:297-301 (1992)

Sera From Simian Immunodeficiency Virus-Infected Rhesus Macaques Inhibit Lymphocyte Proliferation Russell H. Tomar,’12 Paul Hinds,’ David Katz,’ and Kevin S ~ h u l t z ~ ~ ~ Department of Pathology and Laboratory Medicine, and Department of Medicine,2 University of Wisconsin Hospital and Clinics, School of Veterinary M e d i ~ i n eand , ~ Wisconsin Regional Primate Research Centec4 Madison, Wisconsin Rhesus macaque monkeys infected with the simian immunodeficiency virus (SIV) develop a syndrome mimicking acquired immunodeficiency syndrome (AIDS) in humans. We had demonstrated previouslythat sera from individuals infected with human immunodeficiency virus (HIV) inhibit the proliferation of lymphocytes from healthy noninfected subjects and that this phenomenon was associated with the development of clinical AIDS. Thus, we sought to determine whether sera from SIV-infected monkeys would also inhibit lymphocytes from healthy humans and SIV-negative rhesus monkeys. Sera from SIV-infectedmonkeyswere comKey words:

pared with sera from uninfected animals and cultured with cells from healthy human volunteers or SIV-negative monkeys in the presence or absence of phytohemagglutinin (PHA). Cell proliferationwas determined by measuring the incorporation of radiolabeledthymidine into cellular DNA. Sera from SIV-infected monkeys suppressed the proliferationof human and non-humanprimate lymphocytes. This activity appears to be similar to that described for sera from HIV1-infected humans. Therefore, rhesus macaques infected with SIV providea model for the study of serum inhibitory factors previously reported in AIDS patients. o 1992wiley-Liss,IN.

inhibition, AIDS, SAIDS, immune, HIV

INTRODUCTION Sera from humans infected with human immunodeficiency virus type 1 (HIV-1) suppress lymphocyte proliferation in vihp (1-3). Suppression is most evident when infected subjects develop signs and symptoms associated with acquired immunodeficiency syndrome (AIDS); this phenomenon is predictive of disease progression (3). Suppression of cell proliferation is not reversible with I L 2 and occurs at an early stage of lymphocyte stimulation. There appears to be more than one suppressor; one of these is reversible by cyclooxygenase inhibitors whereas the other(s) are not reversible by this drug ( 2 ) . Certain Old World monkeys such as macaques develop an AIDS-like disease when infected with simian immunodeficiency virus (SIV), and thus provide an excellent animal model to study the pathogenesis of such retroviruses. We further explored this model and extended our investigations into suppressor factors by determining whether the sera of animals infected with SIV also suppressed the proliferation of human and non-human primate lymphocytes.

Corb, Delta Regional Primate Center). Blood samples were drawn at 6-7 weeks post-inoculation. Control sera were obtained from two adult rhesus males which had been found negative for SIV, type D and human T-cell leukemia virus type 1 (HTLV-1) retroviruses (4) by enzyme-linked immunosorbent assay (ELISA) and Western blot. Serum samples were aliquoted (0.5- 1.O ml) and stored at - 70°C.

Cells Human blood was obtained by venipuncture from healthy human volunteers not known to be in high risk groups for HIV infection. Mononuclear cells were collected on ficollhypaque gradients (Ficoll-Paque, Pharmacia Fine Chemicals, Piscataway, NJ), washed twice in sterile phosphate buffered saline (pH 7.410.15 M), and suspended in newborn calfenriched culture medium (described below). Simian cells were similarly prepared.

MATERIALS AND METHODS Subjects One female and one male 4-month-old rhesus macaques were inoculated with SIVdelta(courtesy of Dr. M. Murphey0 1992 Wiley-Liss, InC.

Received February 28, 1992; accepted April 10, 1992. Address reprint requests to Dr. Russell H. Tomar, Division of Laboratory Medicine, University of Wisconsin Hospital and Clinics-B4/249,600 Highland Avenue, Madison, WI 53792-2472.

298

Tomar et al.

Tracer Tritiated thymidine [me t h~l -~H] (6.7 Ci/mmol, Dupont NEN, Boston, MA) was diluted with RPMI media (below) to 1 pCi/50 p1 and stored at 3-5°C.

Phytohemagglutinin Purified phytohemagglutinin (PHA) (product HA 16, Wellcome Diagnostics, Research Triangle Park, NC) was reconstituted to 1 mg/ml and then diluted to various concentrations in RPMI 1640 culture medium. In testing simian cells (see Table 5 ) , we reconstituted Bacto PHA-M (Difco Laboratories, Detroit, MI) and then diluted it 1:25 with RPMI 1640.

Cultures RPMI 1640 (GIBCO BRL, Gaithersburg, MD) was supplemented with 5 ml(200 mM) Lglutamine (Sigma Biochemicals, st. Louis, MO), 50,000U penicillin-50 mg streptomycin (ICN-Flow Laboratories, Costa Mesa, CA), and 10% (50 ml) newborn calf serum (Sigma Biochemicals) per 500 ml. Samples were plated in triplicate in flat-bottom microtiter plates (Coming Glass Works-Science Products Division, Coming, NY). Each parameter assayed included both stimulated (with PHA) and unstimulated replicates. Unless otherwise noted, 100,000human mononuclear cells were cultured per well in a total volume of 250 p1 with 2.5% monkey serum, 0.25 pg PHA, plus the supplemented RPMI 1640 described above. After 72-80 hr incubation at 37°C in 5% CO2, 50 ~1 of tritiated thymidine was added to each well. The microplate was returned to the incubator and kept at 37°C for 12- 18 hr. Wells were emptied with a 24 well PHD harvester (Cambridge Technology, Watertown, MA) onto glass fiber filter paper (Cambridge Technology), washed with deionized water 3-4 times followed by 70% ethanol. Punched disks were placed into 7 ml polyethylene scintillation vials (VWR, Chicago, IL) and 3.5 ml of high-efficiency “Scintisol” cocktail (National Diagnostics, Manville, NJ) was added. Vials were counted in a Beckman model LS5800 L.S.C. (Beckman Instruments, Fullerton, CA).

TABLE 1. Serum From a SIV-Infected Monkey Inhibits Lymphocyte Proliferation’ cpm(mean

?

SD)b

Serum source

Background

PHA

Healthy monkey SIV-infected monkev

758 f 43 775 f 200

71,447 t 2,744 21,959 2 1,554

Net cpm % Inhibition 70,689 21,184

70.0

alOO,OOO cells from a healthy human donor were cultured with 2.5% simian serum in a total volume of 250 kl in the presence of the PHA.

bMean ? standard deivation of replicate (3) wells.

I

d3

I

I

4

d4

d5

1

d6

’ d4

I

1

d6

d5

Incubation Time (days)

Fig. 1. The duration of cultures had little effect on the relative inhibition by SIV( + ) serum. Two representative experiments are shown. Cells were cultured with serum from either healthy or SIV( + ) animals in the presence or absence of PHA. Replicate cultures were harvested on the days denoted on the x axis and net cpm determined (see Materials and Methods).

Calculations Net counts per minute (net cpm) were obtained by subtracting the mean counts per minute of background cultures (which received culture medium without PHA) from the mean counts per minute of the stimulated cultures. We set the net cpm of control sera as “100% proliferation” and compared net cpm of test sera to that standard. Percent inhibition was calculated by subtracting the percent proliferation of each test serum from 100%. Each experiment was repeated at least twice and representative data are displayed in the tables and figures.

RESULTS Table 1 illustrates a typical experiment in which SIV( +) serum profoundly inhibits lymphocyte proliferation. In 25 such experiments under the standard conditions described in Materials and Methods, the range of inhibition was 9-100% with a mean of 61 f 29% and a median of 65%. Only 3 of 25 demonstrated inhibitions of less than 25% (9, 11, and 24%). We cultured cells for 3-6 days to ensure that SIV( ) serum was not delaying cell proliferation. Figure 1 shows that at all times studied, proliferation in SIV( +) serum is suppressed compared with growth of cells in healthy monkey serum. In three of four experiments the amount of inhibition was not affected by the day of harvest. This was similar to data obtained in the past using human HIV( + ) serum (1). One experiment, shown on the right in Figure 1, demonstrated increasing inhibition with time of incubation. Our experience with HIV indicated that the concentrations of cells, serum, and mitogen were critical in detecting inhibition. This was also the case with SIV as shown in Table 2; i.e., an appropriate ratio of serum to cells and a suboptimal (for promoting proliferation) amount of PHA improved the sensitivityof the assay. The inhibitory effect of serum increased as more serum was used. However, as we found with HIV( + )

+

SIV Sera Inhibit Proliferation TABLE 2. Relation of Serum and Mitogen Concentrationsto Inhibition' Serum concentration Serum

(%)

Healthy monkey

1.25

PHA (p,g/ml)

Net cpm

% Inhibition

49,923 114,260 149,493 148,327 51,526 97,881 130,181 130,641 39,738 91,409 135,888 152,130 16,471 44,202 57,990 109,301 6,855 27,191 62,758 107,567 695 7,209 22,996 65,957

-

,125 ,250

,500 1.OOO .I25 ,250 ,500

2.50

1.OOO

5.00

,125 ,250

.500 1.OOO ,125 ,250 ,500

1.25

SIV-infected monkey

1.OOO

,125 .250 ,500

2.50

1.OOO

5.00

,125 .250

,500 1.OOO

-

-

-

65 61 61 26 85 72 51 17 96 91 82 56

alOO,OOOcells from a healthy human donor were incubated, with the serum type and amount as listed, in the absence or presence of PHA at the concentration given. The total well volume was 250 pl and cultures were harvested after 3 days of incubation. Net cpm = mean cpm with PHA - mean cpm without PHA.

sera, the inhibitory effect of SIV( +) sera can be overcome by increasing the quantity of mitogen. These effects were reproduced in three separate experiments and only a representative one is shown in Table 2 . The factor(s) in SIV( ) serum responsible for inhibition

+

Leneth of Cultyce .......g ........ 120 Hours 1 2 m

-

could be diluted out with serum from a healthy macaque (Fig. 2 ) . This plus the data in Table 2 illustrating that 1.25% serum from a healthy animal could support lymphocyte proliferation demonstrates that SIV( +) serum is not missing nutrients necessary for proliferation but is indeed inhibitory. We carried out a series of experiments to determine how early in culture SIV( +) serum needed to be present to suppress proliferation. Cultures were established with serum from healthy or infected monkeys (0 time). The cultures were centrifuged and the supernate removed and replaced by media with SIV( +) or healthy serum at predetermined time intervals. One of two such experiments is shown in Table 3 and its data suggest that SIV( +) serum had to be present early during incubation with PHA to be suppressive. Cyclooxygenase inhibitors partially reverse the inhibitory effects of HIV( +) serum. In three separate experiments, we added indomethacin to cells cultured with SIV( + ) serum to determine if a similar effect could be seen in the simian system. A representative result is shown in Table 4 demonstrating that indomethacin, a cyclooxygenase inhibitor, changed the inhibition from 39 to 24 and 3%, respectively. Since all of the above were performed using human cells, we sought to determine if HIV( +) and SIV( + ) sera were suppressive to monkey lymphocytes. Peripheral blood cells from a healthy Macaca mulatta were separated on ficollhypaque gradients and cultured with or without PHA (Difco 1:25) in the presence of healthy monkey serum, healthy human serum, HIV( ), or SIV( + ) serum. Results are shown in Table 5. Both HIV( +) and SIV( + ) sera inhibit the proliferation of PHA-driven simian lymphocytes.

+

DISCUSSION The disease induced in rhesus macaques by SIV resembles AIDS sufficiently to provide investigators with an excellent model of HIV-1 infection (5-8). In this manuscript, we describe one more similarity. The only difference between HIV( + ) and SIV( + ) serum-induced suppression is that SIV( ) serum appears to be more potent. Two major ques-

+

+96HOvrs

TABLE 3. Effect of Adding Serum During the Course of Incubation'

..... ........

E a

2 z"

299

...... smw:

:

100

80

6a

20

40

90Normal Monkey Serum

Fig. 2. The inhibitory effect of SIV( + ) serum could be titered with serum from uninfected animals. Two representative experiments are shown in which cells were cultured in serum from healthy or SIV( ) animals and in various mixtures of the two.

+

Simian serum (net cpm)

Hours of incubation

Healthy

SIV-infected

% Inhibition

Initiation (0) 1 4 25

132,479 142,403 137,740 162.139

55,495 81,762 135,747 159.623

58 42 1 1

"100,OOO cells from a healthy human donor were cultured with serum from a healthy animal with or without PHA. At the times given, the cultures were removed from the incubator, centrifuged, and the supernate removed and replaced with media containing healthy (controls) or SIV-infected serum. All cultures were harvested after 4 days of incubation. Net cpm-mean cpm with PHA -mean cpm without PHA (background).

300

Tomar et al.

TABLE 4. Partial Reversal of Inhibition by Indomethacina Simian serum

Indomethacin

Net cpm

Healthy SIV-infected Healthy SIV-infected Healthy "infected

-

189,555 115,265 194,075 146,900 183,090 177.763

-

1:1,OOo 1:1,OOo 1:2,000 1:z.OOo

%Inhibition

39 -

24 -

3

"100,OOO cells from a healthy human donor were cultured with or without PHA in the presence or absence of indomethacin (1 mg/ml stock) and harvested after 4 days of culture.

tions remain unanswered: what are these inhibitory substances and what role, if any, do they play in the development of disease. Our work with HIV-1( ) serum indicates that there are at least three inhibitory materials (2). This was based on patterns of suppression of lymphocytic cell lines where all the AIDS sera tested inhibited lymphocytes from healthy individuals and thus were considered to be suppressive. However, different patterns were observed with cell lines. Some AIDS sera inhibited one set of cell lines but not another; others inhibited the second set but not the first; a third series of sera inhibited both cell lines; and a fourth collection of sera inhibited neither set of cell lines. Suppression of proliferation of one set was partially reversible through the addition of cyclooxygenase inhibitors such as indomethacin or ibuprofen (2). Indeed the suppression caused by most sera using peripheral blood lymphocytes from healthy donors is partially reversed by the same agents. The two most likely sources of suppression are viral products or host-derived cytokines. Whole virus or various components of HIV-1 have been shown to have effects on lymphocyte proliferation (8- 15). Certainly any one or a combination of these in the serum could have an inhibitory effect on lymphocyte proliferation. The serum suppressive effect peaks at a time when the viral load appears to be increasing

+

TABLE 5. Inhibition of Simian Cells by SIV( +) and HIV( +) Seraa PHAb Serum source Healthy monkey SIV-infected Healthy human HIV-infected

1 :25(n) -3

+2 -3 +3 -3 +2 -3 +2

CPm (mean i SD)

307 i286 14,315i 1,377 252 i 298 6,185? 4,406 204i 312 13,456i 284 207 i 143 3.235? 1.705

Net cpm

% Proliferation

14,007

100

5,933

42

13,252

100

3.029

23

"100,OOocells from a healthy rhesus macaque were cultured with healthy monkey, healthy human, SIV( +), or HIV( +) sera in the presence or absence of PHA. Cultures were harvested after 4 days. bBacto PHA-M diluted 1:25with RPMI 1640 medium.

(3,16). Since viral load is also increased early after infection we would expect to see serum suppression at that time as well. To date we have not found suppression early, although it is possible that we missed the early viremia. We are in the process of studying serum samples at different times after infection with SIV. It is unlikely that replicating virus plays any role in the phenomenon we are studying since we pretreat sera at 56°C for 30 min prior to each assay. Cytokines also influence lymphocyte function and IL2 (17, IL-6 (18), interferons (19), and TGF-beta (20) levels have already been shown to be affected by infection with HIV- 1 and cytokines affect HIV-1 production (21). It is conceivable that one or more cytokines might be increasingly produced during infection until a level is achieved to cause inhibition of lymphocyte multiplication. Teleologically, one might speculate that such an inhibitory substance might protect the host by dampening viral replication which is accelerated in activated lymphocytes. Since there appears to be more than one factor, these possibilities are not mutually exclusive. There is evidence that these factors are associated with the progression of infection to disease (3). We compared several serum factors with CD4+ lymphocyte levels among healthy controls who were HIV( - ), HIV( - ) subjects with increased risk factors for AIDS, HIV( + ) asymptomatic subjects, HIV( + ) subjects with symptoms but not AIDS, and persons with AIDS. Inhibition of lymphocyte proliferation nearly equaled absolute CD4 levels in predicting the appropriate classification (3). The presence of the suppressive phenomenon in monkeys will allow the timing of appearance of suppression to be answered and provide sufficient quantities of serum to allow further characterizationof the inhibitory substance. We believe that study of serum suppression may provide further understanding of the pathogenesis of SAIDS and AIDS.

ACKNOWLEDGMENTS This work was supported in part by NIH RR 00167 to the Wisconsin Regional Primate Research Center (WRPRC) and the School of Veterinary Medicine. This is publication no. 3 1-004 from the WRPRC.

REFERENCES Hennig AK, Tomar RH: Inhibition of in vitro lymphocyte proliferation by serum from acquired immune deficiency syndrome patients depends on the ratio of cells to serum in culture. Clin Immunol Immunopathol 33:258-267,1984. Hennig AK, John PA, Blair DC, Tomar RH: Sera from AIDS patients contain multiple factors that suppress in vitro proliferation of lymphoid cells. JClin Lab Anal 2:215-219, 1988. Tomar RH, Hennig AK, Oates RP, Yuille MA, John PA: Serum factors in the progression of human immunodeficiency virus type 1 infection to AIDS. J Clin Lab Anal 4218-223, 1990. Lairmore MD, Lerche NW, Schultz K, Stone CM, Brown BG, Hermann LM, Yee JA, Jennings M: SIV, STLV-1, type D retrovirus antibodies in captive rhesus macaques and immunoblot reactivity to SIV

SIV Sera Inhibit Proliferation

5. 6.

7.

8.

9.

10.

11.

12.

13.

p27 in human and rhesus monkey sera. AIDS-Res Hum Retrovir 6: 1233-1238, 1990. Berzofsky JA: Approaches and issues in the development of vaccines against HIV. JAcquirImmune Defic Syndr 4:451-459, 1991. King NW, Letvin NL: Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques. JAcquirImmune DeficSyndr 3:1023-1040, 1990. Chakrabarti L, Guyader M, Alizon M, et al.: Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature 328543-547. 1987. Arya SK, Beaver B, Jagodzinski L, et al.: New human and simian HIVrelated retroviruses possess functional transactivator (tat) gene. Nature 3281548-550, 1987. Hofmann B, Nishanian P, Baldwin RL, Insixiengmay P, Nel A, Fahey JL: HIV inhibits the early steps of lymphocyte activation, including initiation of inositol phospholipid metabolism. J Immunol 121:36993705, 1990. Hofman B, Langhoff E, Lindhardt BO, et al.: Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera. Cell Immunol12 1:336-348, 1989. Krowka J, Stites D, Debs R, Larsen C, Fedor J, Brunette E, Duzgunes N: Lymphocyte proliferative responses to soluble and liposome-conjugated envelope peptides of HIV-I. JImmunol144:2535-2540, 1990. Viscidi RP, M a y K, Lederman HM, Frankey AD: Inhibition of antigeninduced lymphocyte proliferation by tat protein from HIV-I . Science 246:1606-1608, 1989. Weinhold KJ, Lyerly HK, Stanley SD, Austin AA, Matthews TJ, Bolognesi DP: HIV-1 GP120-mediated immune suppression and lymphocyte destruction in the absence of viral infection. J Immunol 142:3091-3097. 1989.

301

14. Kornfeld H, Cruikshank WW, Pyle SW, Berrnan JS, Center DM: Lymphocyte activation by HIV- 1 envelope glycoprotein. Nature 335: 445-448, 1988. 15. Pahwa S , Pahwa R, Saxinger C , Gallo RC, Good RA: Influence of the human T-lymphotropic virus/lymphadenopathy-associatedvirus on functions of human lymphocytes: Evidence for immunosuppressive effects and polyclonal B-cell activation by banded viral preparations. Proc Natl Acad Sci USA 82:8198-8202, 1990. 16. Ho DD, Moudgil T, Alan M: Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med 321: 1621- 1625, 1989. 17. Tsunetsugu-Yokota Y,Honda M: Effect of cytokines on HIV release and IL-2 receptor alpha expression in monocytic cell lines. J Acquir ImmuneDeficSyndr 3 5 1 1-516, 1990. 18. Breen EC, Rezai AR, Nakajima K, Beall GN, Mitsuyasu RT, Hirano T, Kishimoto T, Martinez-Mazo 0: Infection with HIV is associated with elevated I L 6 levels and production. J lmmunol144:480-484, 1990. 19. Kmwn SE, Niedzwiecki D, Bhalla RB, Flomenberg N, Bundow D, Chapman D: Relationship and prognostic value of endogenous interferonalpha, beta 2-microglobulin, and neopterin serum levels in patients with Kaposi sarcoma and AIDS. J Acquir Immune Defic Syndr 4: 871-880,1991. 20. Kekow J, Wachsman W, McCutchan JA, Cronin M, Carson DA, Lotz M: Transforming growth factor p and noncytopathic mechanisms of immunodeficiency in human immunodeficiency virus infection. Proc Natl AcadSci USA 87:8321-8325, 1990. 21. Koyangi Y, O’Brien WA, Zhao JQ, Golde DW, Gasson JC, Chen ISY: Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science 241 :1673- 1675, 1988.