Clin. exp. Immunol (1991) 83, 192-196

ADONIS 000991049100036P

Effect of a recombinant HIV gpl60 vaccine on monokine production D. P. DOOLEY*t, R. A. COX* & D. J. LOONEYJ *Department of Research Immunology, San Antonio State Chest Hospital, San Antionio, TX, tInfectious Disease Service, Brooke Army Medical Center, Fort Sam, Houston, TX, and JDivision of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD, USA

(Acceptedfor publication 6 August 1990)

SUMMARY An investigation was undertaken to determine whether a recombinant gp 160 envelope protein, which is currently being evaluated as a vaccine for AIDS, induces or modulates the production of tumour necrosis factor-alpha (TNF-a) or interleukin-l (IL-If. Incubation of monocytes from healthy, HIV-seronegative persons with 0 0001-1 0 ug of the recombinant vaccine did not result in the secretion of TNF-a or IL-I#, nor did the recombinant product augment or suppress monokine production by lipopolysaccharide (LPS) stimulated monocytes. The vaccine was also without a stimulatory or modulatory effect upon TNF-a or IL-IfP secretion by monocytes from a patient with the AIDS-related complex (ARC) and from the monocytic THP-l cell line. The lack of effect of gp 160 on monokine production has important implications for its efficacy as a vaccine for AIDS. Keywords AIDS monokines gpl6O interleukin-l tumour necrosis factor-alpha

INTRODUCTION Tumour necrosis factor-alpha (TNF-a) and interleukin- lIl (IL-If?) possess protean immunomodulatory, inflammatory, and metabolic effects (reviewed by Dinarello, 1984; Le & Vilcek, 1987; Beutler & Cerami, 1989). Cumulative studies have provided evidence that these monokines are produced in response to infection with HIV. Serum levels of TNF-ot and IL- IB have been reported to be elevated in patients with AIDS (Lahdevirta et al., 1988; Reddy et al., 1988; Hober et al., 1989; Lepe-Zuniga, Mansell & Hersh, 1987; Reddy & Grieco; 1989), and monocytes/macrophages from patients with progressive HIV infection have been shown to secrete high levels of TNF-a and IL-lI#, either constitutively or following stimulation with lipopolysaccharide (LPS) (Eden & Turino, 1986; Haas, Riethmuller & Ziegler-Heitbrock, 1987; Berman et al., 1987; Wright et al., 1988; Roux-Lombard et al., 1989; Lau & Livesey, 1989; Hober et al., 1989; Weiss et al., 1989; Cox et al., 1990). The possibility that the elevated monokine production observed in HIV-infected persons is elicited by the virus itself is supported by the finding that normal blood monocytes or monocytic cell lines infected in vitro with HIV elaborate increased levels of TNF-cx and IL-I# (Molina et al., 1989; Merrill, Koyanagi & Chen, 1989; Vyakarnam et al., 1990). The induction of TNF-a and IL-13 could have important consequences on the course of HIV infection. These monokines may augment host defence by activating immune effector cells (Dinarello, 1984; Le & Vilcek, 1987; Beutler & Cerami, 1989);

however, they may be detrimental to the host by inducing acute inflammatory processes that can lead to tissue damage or, with TNF-a, the production of cachexia (Beutler & Cerami, 1989). Moreover, recent reports have established that TNF-a and IL- 1 can potentiate the replication of HIV in infected lines (Koyanagi et al., 1988; Clouse et al., 1989a, 1989b; Duh et al., 1989; Folks et al., 1989; Matsuyama et al., 1989; Okamoto et al., 1989; Osborn, Kunkel & Nabel, 1989; Israel et al., 1989; Kobayashi et al., 1989). The pleiotrophic effects of TNF-a and IL-if and their production during HIV infection warrant analyses to determine whether vaccines developed for this disease might induce or modulate these monokines. Here we examined the effect of a recombinant gp 160 envelope vaccine, which is currently being evaluated in clinical trials of healthy, HIV-seronegative persons (Barnes, 1987; Kovacs et al., 1988; Dolin et al., 1989; Keefer et al., 1989; Koff & Fauci, 1989). MATERIALS AND METHODS

Reagents The recombinant HIV gpl60 envelope glycoprotein was developed by MicroGeneSys (West Haven, CT) and was provided under the direction of the Walter Reed Retrovirus Research Group (Rockville, MD). This recombinant product was free of detectable LPS as determined by a chromogenic Limulus amoebocyte lysate test (M.A. Bioproducts, Walkersville, MD). The monocytic leukaemia THP- I cell line was obtained from the American Type Culture Collection (ATCC no. TIB 202; Rockville, MD) and maintained by passage in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf

Correspondence: Dr Rebecca Cox, Research Immunology, San Antonio State Chest Hospital, San Antonio, TX 78223, USA.

192

193

Monokine induction by gpJ60 (GIBco) and 2 x 10-5 M 2-mercaptoethanol. LPS from Escherichia coli serotype 0127: B8 was obtained from Sigma Chemical Company (St Louis, MO). Murine monoclonal OKT4A was obtained from Ortho Diagnostics Systems (Raritan, NJ).

nr

serum

Isolation of blood monocytes Venous blood from healthy, HIV-seronegative volunteers was collected in heparinized tubes (20 U heparin/ml). Mononuclear cells were isolated by gradient centrifugation on FicollHypaque (Pharmacia Fine Chemicals, Piscataway, NJ) and washed twice in RPMI 1640 medium containing 2 mm glutamine, streptomycin (100 pg/ml), penicillin (100 U/ml), and 5% human AB serum (Sigma) (hereafter designated supplemented RPMI). The cells were suspended to 2 x 106/ml of medium, and I0-ml aliquots were incubated on plastic Petri dishes (15-cm diameter) for 1 h at 370C under a humidified 5% CO2 environment. Non-adherent cells were removed by washing. Warm RPMI 1640 was added to the Petri dishes, and the adherent cells were again incubated for 1 h at 370C. The cells were harvested by gently scraping with a cell scraper in cold medium and suspended to 2 x 106/ml of supplemented RPMI. Viability was consistently greatly than 92%, as assessed using trypan blue dye exclusion, and at least 70% of the adherent cells were monocytes, as judged by non-specific esterase staining (Koski, Poplack & Blaese, 1986). Induction of monokines Peripheral blood (0-1 ml) mononuclear cells (2 x I05/ml) or THP- 1 cells (5 x 105/ml) were dispensed to wells on a 96-well round-bottomed microtitre plate (Corning Glass Works, Corning, NY). The cells were cultured with various concentrations of recombinant gpl60 alone, LPS alone, or gpl60 followed by LPS. Negative control wells received medium alone. After overnight incubation at 37°C under 5% C02, supernatants were collected by centrifugation and stored at 70°C until assayed. -

Monokine assays Supernatants were assayed for TNF-a using an ELISA obtained from Endogen (Boston, MA). IL-lfl was quantified using an ELISA kit purchased from Cistron Biotechnology (Pine Brook, NJ). The sensitivities of the ELISA procedures were 60 pg/ml for TNF-a and 20 pg/ml for IL-1,B (manufacturers' inserts).

RESULTS Effects of gpl60 on monokine production by blood monocytes The TNF-a responses obtained when normal monocytes were incubated with gpl60 alone or in combination with LPS are shown in Fig. 1. Recombinant gpl60, at a concentration of 1 ug, did not directly stimulate the secretion of TNF-a within an 18-h exposure period. This lack of a direct stimulatory effect was also observed when monocytes were incubated with lower concentrations of gpl60, ranging from 0-0001 to 0 50 pg. Concentrations above 1 pg were toxic to macrophages and were not evaluated beyond preliminary assays. To determine whether gpl60 modulates the stimulatory effect of LPS, monocytes were pre-incubated for 1 h with gp 160 and then cultured for 18 h with LPS. As shown in Fig. 1, the recombinant product neither augmented nor suppressed TNF-a production by LPS-stimulated monocytes. This lack of a

8

,E>

6

0'

0

z

2

I

a

a

None

gp

160

LPS

I

9

gp

160 +

LPS

Fig. 1. Secretion of tumour necrosis factor-alpha (TNF-a) by blood monocytes from healthy, HIV-seronegative donors. Monocytes (2 x 105) were incubated with gpl60 alone; lipopolysaccharide (LPS) alone; or gpl60 for lh followed by LPS. LPS and gpl60 were each used at a concentration of 1 pg per 2 x 105 cells. Supernatants were collected 18 h later and assayed for TNF-a as described. Results are mean + s.e.m. of responses obtained in assays of four donors.

modulatory effect was also observed when the concentrations of gpl60 and LPS were varied (data not shown) and when monocytes were pre-incubated with gpl6O for various time periods before addition of LPS (Fig. 2). Merrill et al. (1989) reported that HIV triggers monocytes to produce TNF-c by binding to the CD4 receptor. Monokine secretion is similarly induced by the interaction of monoclonal OKT4A with CD4 (Merrill et al., 1989; Wahl et al., 1989). In a comparison of monokine induction by OKT4A and gpl 60, we detected 4436 pg of TNF-ca per ml of supernatant from monocytes stimulated with 5 pg of OKT4A, and no TNF-a in supernatants from gpl6O-stimulated monocytes. The IL-1 responses of monocytes incubated with gpl6O alone or in combination with LPS are shown in Fig. 3. Concentrations of gpl60 ranging from 0-0001 to 1 0 pg did not induce detectable levels of IL-I, and were without an effect on the response of the monocytes to LPS. The preceding studies were performed using monocytes from HIV-seronegative donors. To determine whether monocytes from an HIV-infected person might be more responsive to modulation by gpl6O, we examined monokine production by cells from a patient with ARC. The recombinant protein did not stimulate the patient's monocytes to secrete TNF-a or IL-#, and gpl 60 did not affect the response of LPS-stimulated monocytes from this patient (Table 1).

Effect of gpl60 on monokine production by THP-J cells Studies were next conducted to determine whether gp 160 induces or modulates monokine production by the human

194

D. P. Dooley, R. A. Cox & D. J. Looney 20

Table 1. Effect of gpl60 on tumour necrosis factor-alpha (TNF-a) and interleukin-lfl (IL-IfJ) production by blood monocytes from a patient with ARC

15

Monokine response (pg/ ml)

CY'>

Stimulus

TNF-a

IL-flf

56 44 2520 2388

0 0 596 620

0

None gpl60 LPS gpl6O+LPS

0

z

0

2

4 Time (h)

6

i

18

Fig. 2. Kinetics of secretion of tumour necrosis factor-alpha (TNF-a) by blood monocytes from a healthy, HIV-seronegative donor. Monocytes (2 x 105) were incubated with lipopolysaccharide (LPS) alone (closed circles) or with gpl60 for I h followed by LPS (open circles), each at a concentration of 1 Mg/2 x 105 cells. Supernatants were collected 2, 4, 6, and 18 h later and assayed for TNF-a as described. Responses obtained in a representative experiment.

Blood monocytes (2 x 105) were cultured for 18 h with medium alone or medium containing gp160 or lipopolysaccharide (LPS), each at a final concentration of 1 pg. The effect of gp 160 on the response to LPS was assessed by pre-incubating monocytes with gpl60 (1 pg) for 1 h, and then adding LPS (1 pg) and incubating the cultures for 18 h.

Table 2. Effect of gp 160 on tumour necrosis factor-alpha (TNF-a) and interleukin-l13 (IL-1I#) production by THP-1 cells Monokine response

(pg/ml)

10 Stimulus None

gpl60 LPS gpl6O+LPS

6E 6

TNF-a

IL-1fl

0 0 413 308

0 0 240 288

THP-1 cells (5 x 105) were cultured for 18 h with medium alone or medium containing gp 160 or lipopolysaccharide (LPS), each at a final concentration of 1 pg. The effect of gp 160 on the response to LPS was assessed by pre-incubating THP- I cells with gp 160 (1 pg) for 1 h, and then adding LPS (1 pg) and incubating the cultures for 18 h.

0

74

2

None

gp 160

LPS

gp 160 +

LPS

Fig. 3. Secretion of interleukin-lfl (IL-I/3) by blood monocytes from healthy. HIV-seronegative donors. Monocytes (2 x 105) were incubated with gpl6O alone; lipopolysaccharide (LPS) alone; or gpl6O for 1 h followed by LPS. LPS and gpl6O were each used at a concentration of I pg per 2 x 105 cells. Supernatants were collected 18 h later and assayed for IL- 1# as described. Results are mean + s.e.m. of responses obtained in assays of six donors.

monocytic leukaemia THP- I cell line, a cell line which has been reported to secrete TNF-cx and IL-I3 during acute-phase infection with HIV (Molina et al., 1989). As shown in Table 2, THP-1 cells did not elaborate TNF-a or IL-I# in response to gp 160 and the recombinant protein was without a demonstrable effect on LPS-induced monokine secretion by THP-I cells. Since CD4 receptor expression may decrease upon culture (Kazazi et al., 1989), assays were performed to establish that CD4 receptors were expressed on the THP- I monocytic cell line. Indirect immunofluorescence using murine monoclonal antiOKT4 revealed that 92% of the THP-l cells were CD4 positive.

Monokine induction by gpJ60 DISCUSSION The results of this study provide evidence that a recombinant HIV envelope gp160 protein does not induce monocytes/ macrophages to secrete TNF-a or IL- I f, and does not modulate the production of these monokines by LPS-stimulated monocytes/macrophages. The lack of effect of the recombinant product was observed at a wide range of conditions in which we evaluated gradient concentrations of gpl60, using non-stimulated and LPS-stimulated monocytes/macrophages from HIVseronegative persons, a patient with ARC, and the THP-l cell line. Studies on the induction of monokines by HIV-derived envelope proteins have been limited to two reports. Wahl et al. (1989) found that immunoaffinity-purified native gpl 20 induced IL-l secretion by normal human blood monocytes. In contrast, Molina et al. (1990) did not detect the production of IL-If? or TNF-a by normal monocytes incubated with a recombinant gp1 20 product. The diversity in these results could be attributed to compositional or structural differences in the native and recombinant gpl20 preparations, or to the use of freshly isolated monocytes versus 9-day-old adherent cells in the studies by Wahl et al. (1989) and Molina et al. (1990), respectively. During the initial phases of the present investigation, we evaluated TNF-a and IL-ifl production by freshly isolated monocytes incubated with an immunoaffinity-purified gpl20 (provided by Dr Larry 0. Arthur, Frederick Cancer Research Facility, Frederick, MD). Native gpI20 induced significant levels of TNF-a and IL-if? (data not presented). However, the gpl20 preparation was positive for LPS when tested with the Limulus lysate reagent, and it was ineffective in eliciting monokines when assayed in the presence of polymyxin B, indicating that the monokine responses were attributed to LPS and not gpI60. It is noteworthy that although the immunoaffinity-purified gpl2O used by Wahl et al. (1989) was reported to contain less than 1 ng of LPS/ml, LPS is a potent inducer of monokines, even at pg levels, and it is possible that the level of LPS contamination was sufficient to account for the monokine responses observed by those investigators. Divergent results have also been obtained in investigations of monokine induction by live or inactivated HIV. Molina et al. (1989) reported that HIV did not directly induce TNF-a or IL-Ilf in THP-I monocytic cells, but did augment the response of the cells to LPS stimulation, an effect that was observed in acutely, but not chronically infected monocytes. However, Merrill et al. (1989) reported that exposure to live HIV directly induced TNF-c and IL-lI# production in non-stimulated blood monocytes. Conversely, Roy et al. (1988) found that in vitro infection of normal human blood monocytes suppressed rather than augmented IL-I production in LPS-stimulated cells; more recently, Munis, Richman & Kornbluth (1990) and Molina et al. (1990) reported that live HIV-I did not augment or suppress IL- I or TNF-ax production by human blood monocyte-derived macrophages. It is clear that additional studies are needed to determine whether HIV modulates monokine responses and, if so, to establish whether monokine responses are also modulated by specific components of the virus. The latter is particularly relevant to assessing the efficacy and safety of vaccines for AIDS. The induction or modulation of monokines by the virus and viral-derived vaccines could have important consequences

195

on the course of HIV infection. At physiologic concentrations, TNF-a and IL-14J can augment the activity of immune effector cells (Dinarello, 1984; Le & Vilcek, 1987; Beutler & Cerami, 1989). However, when produced in exaggerated amounts, as has been observed in some patients with progressive HIV infection (Lahdevirta et al., 1988; Reddey et al., 1988; Wright et al., 1988; Roux-Lombard et al., 1989; Lau & Livesey, 1989; Hober et al., 1989; Cox et al., 1990), TNF-a and IL-1# can produce severe inflammatory reactions and, in response to TNF-a, metabolic perturbations that may be fatal (Dinarello, 1984; Le & Vilcek, 1987; Beutler & Cerami, 1989). The potential deleterious effects of TNF-ox and IL-ifi are further underscored by reports that these monokines promote HIV gene expression in infected cell lines (Koyanagi et al., 1988; Clouse et al., 1989a, 1989b; Duh et al., 1989; Folks et al., 1989; Israel et al., 1989; Matsuyama et al., 1989; Okamoto et al., 1989; Kobayashi et al., 1989). If this augmentation occurs in vivo, the induction of TNF-a or IL-1if by vaccines could adversely modify the course of HIV infection. Thus, our finding that the recombinant gpl6O product neither induces nor modulates monokine production argues in favour of its safety as a vaccine candidate for this disease. Additional work is needed to corroborate these results, with emphasis on the effect of the recombinant product on monocytes from persons with early, asymptomatic HIV infection.

ACKNOWLEDGMENTS We wish to thank Donna Wimberly and Wanda Rasmussen for their expert technical assistance.

REFERENCES BARNES, D.M. (1987) AIDS vaccine trial OKed. Science, 237, 973. BERMAN, M.A., SANDBORG, C.I., CALABIA, B.S., ANDREWS, B.S. & FRIOU, G.J. (1987) Interleukin 1 inhibitor masks high interleukin 1 production in acquired immunodeficiency syndrome (AIDS). Clin. Immunol. Immunopathol. 42, 133. BEUTLER, B. & CERAMI, A. (1989). The biology of cachectin/TNF-a-a primary mediator of the host response. Annu. Rev. Immunol. 7, 625. CLOUSE, K.A., POWELL, D., WASHINGTON, I., POLI, G., STREBEL, K., FARRAR, W., BARSTAD, P., KOVACS, J., FAUCI, A.S. & FOLKS, T.M. (1989a) Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J. Immunol. 142, 431. CLOUSE, K.A., ROBBINS, P.B., FERNIE, B., OSTROVE, J.M. & FAUCI, A.S. (1989b) Viral antigen stimulation of the production of human monokines capable of regulating HIV- 1 expression. J. Immunol. 143, 470. Cox, R.A., ANDERS, G.T., CAPPELLI, P.J., JOHNSON, J.E., BLANTON, H.M., SEAWORTH, B.J. & TREASURE, R.L. (1990) Production of tumor necrosis factor-alpha and interleukin-1 by alveolar macrophages from HIV-1 infected persons. AIDS Res. hum. Retroviruses, 4, 431. DINARELLO, C.A. (1984) Interleukin-l. Rev. infect. Dis. 6, 51. DOLIN, R., GRAHAM, B., GREENBERG, S. & TACKETr, C. (1989) Safety and immunogenicity of HIV- 1 recombinant gp 160 vaccine candidate in normal volunteers. In V. International Conference on AIDS, Montreal, Canada (Abstract Th.C.O.33) p. 538. DUH, E.J., MAURY, W.J., FOLKS, T.M., FAUCI, A.S. & RABSON, A.R. (1989) Tumor necrosis factor a activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. natl Acad. Sci. USA, 86, 5874. EDEN, E. & TURINO, G.M. (1986) Interleukin 1 secretion from human alveolar macrophages in lung disease. J. clin. Immunol. 6, 326.

196

D. P. Dooley, R. A. Cox & D. J. Looney

FOLKS, T.M., CLOUSE, K.A., JUSTEMENT, J., RABSON, A., DUH, E. KEHRL, J.H. & FAUCI, A.S. (1989) Tumor necrosis factor a induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. natl Acad. Sci. USA, 86, 2365. HAAS, J.G., REITHMULLER, G. & ZIEGLER-HEITBROCK, H.W.L. (1987) Monocyte phenotype and function in patients with the acquired immunodeficiency syndrome (AIDS) and AIDS-related disorders. Scand. J. Immunol. 26, 371. HOBER, D., HAQUE, A., WATTRE, P., BEAUCAIRE, G., MOUTON, Y. & CAPRON, A. (1989) Production of tumour necrosis factor-alpha (TNF-a) and interleukin-I (IL-1) in patients with AIDS. Enhanced level of TNF-a is related to a higher cytotoxic activity. Clin. exp. Immunol. 78, 329. ISRAEL, N., HAZAN, U., ALCAMI, J., MUNIER, A., ARENZANA-SEISDEDOS, F., BACHELERIE, F., ISRAEL, A. & VIRELIZIER, J.-L. (1989) Tumor necrosis factor stimulates transcription of HIV- I in human T lymphocytes, independently and synergistically with mitogens. J. Immunol. 143, 3956. KAZAZI, F., MATHIJS, J.-M., FOLEY, P. & CUNNINGHAM, A.L. (1989) Variations in CD4 expression by human monocytes and macrophages and their relationship to infection with the human immunodeficiency virus. J. gen. Virol. 79, 2661. KEEFER, M.C., BONNEZ, W., ROBERTS, N.J. JR, DOLIN, R. & REICHMAN, R.C. (1989) HIV-1 recombinant gpl60 vaccine recipients demonstrate gpl60 specific lymphocyte proliferation prior to Western blot reactivity. In V. International Conference on AIDS, Montreal, Canada (Abstract M.C.P. 8) p. 543. KOBAYASHI, N., MAMAMOTO, Y., KOYANAGI, Y., CHEN, I.S.Y. & YAMAMOTO (1989) Effect of interleukin-1 on the augmentation of human immunodeficiency virus gene expression. Biochem. Biophys. Res. Commun. 165, 715. KOFF, W.C. & FAUCI, A.S. (1989) Human trials of AIDS vaccines: current status and future directions. AIDS, 3, S125. KOSKI, I.R., POPLACK, D.G. & BLAESE, R.M. (1986) A non-specific esterase stain for the indentification of monocytes and macrophages. In In Vitro Methods in Cell-Mediated and Tumour Immunity (Ed. by B. R. Bloom & J. R. David) p 359, Academic Press, New York.

KOVACS, J.A., MEGIL, M.E., DEYTON, L., METCALF, J.A., SALZMAN, N., VASUDE-VACHARI, M., BASELER, M., MARTIN, M., FOLKS, T.M., SMITH, G., VOLVOVITZ, F., MASUR, H., FAUCI, A.S. & LANE, H.C. (1988) Phase I trial of a recombinant gpl 6O candidate AIDS vaccine. In IV International Conference on AIDS, Stockholm, Sweden (Abstract 6569) p. 289. KOYANAGI, Y., O'BRIEN, W.A., ZHAO, J.Q., GOLDE, D.W., GASSON, J.C. & CHEN, I.S.Y. (1988) Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science, 241, 1673. LAHDEVIRTA, J., MAURY, C.P.J., TEPPO, A.-M. & REPO, H. (1988) Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome. Am. J. Med. 85, 289. LAU, A.S. & LIVESEY, J.F. (1989) Endotoxin induction of tumor necrosis factor is enhanced by acid-labile interferon-a in acquired immunodeficiency syndrome. J. clin. Invest. 84, 738. LE, J. & VILCEK, J. (1987) tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab. Invest. 56, 234. LEPE-ZUNIGA, J.L., MANSELL, P.W.A. & HERSH, E.M. (1987) Idiopathic production of interleukin-l in acquired immune deficiency syndrome. J. clin. Microbiol. 25, 1695.

MATSUYAMA, T., YOSHIYAMA, H., HAMAMOTO, Y., YAMAMOTO, N., SOMA, G.-I., MIZUNO, D. & KOBAYASHI, N. (1989) Enhancement of HIV replication and giant cell formation by tumor necrosis factor. AIDS Res. hum. Retroviruses, 5, 139. MERRILL, J.E., KOYANAGI, Y. & CHEN, I.S.Y. (1989) Interleukin-l and tumor necrosis factor-a can be induced from mononuclear phagocytes by human immunodeficiency virus type 1 binding to the CD4 receptor. J. Virol. 63, 4404. MOLINA, J.-M., SCADDEN, D.T., BYRN, R., DINARELLO, C.A. & GROOPMAN, J.E. (1989) Production of tumor necrosis factor a and interleukin 1? by monocytic cells infected with human immunodeficiency virus. J. clin. Invest. 84, 733. MOLINA, J.-M., SCHINDLER, R., FERRIANI, R., SAKAGUCHI, M., VANNIER, E., DINARELLO, C.A. & GROOPMAN, J.E. (1990) Production of cytokines by peripheral blood monocytes/macrophages infected with human immunodeficiency virus type 1 (HIV-1). J. infect. dis. 161,888. MUNIS, J.R., RICHMAN, D.D. & KORNBLUTH, R.S. (1990) Human immunodeficiency virus-I infection of macrophages in vitro neither induces tumor necrosis factor (TNF)/cachectin gene expression nor alters TNF/cachectin induction by lipopolysaccharide. J. cdin. Invest. 85, 591. OKAMOTO, T., MATSUYAMA, T., MORI, S., HAMAMOTO, Y., KOBAYASHI, N., YAMAMOTO, N., JOSEPHS, S.F, WONG-STAAL, F. & SHIMOTOHNO, K. (1989) Augmentation of human immunodeficiency virus type 1 gene expression by tumour necrosis factor-a. AIDS Res. hum. Retroviruses, 5, 131. OSBORN, L.S., KUNKEL, S. & NABEL, G.J. (1989) Tumor necrosis factora and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. natl Acad. Sci. USA, 86, 2336. REDDY, M.M., SORRELL, S.J., LANGE, M. & GRIECO, M.H. (1988) Tumor necrosis factor and HIV p24 antigen levels in serum of HIVinfected populations. J. AIDS, 1, 436. REDDY, M.M. & GRIECO, M.H. (1989) Neopterin and alpha and beta interleukin- 1 levels in sera of patients with human immunodeficiency virus infection. J. clin. Microbiol. 27, 1919. ROUX-LOMBARD, P., MODOUX, C., CRUCHAUD, A. & DAYER, J.-M. (1989) Purified blood monocytes from HIV 1-infected patients produce high levels of TNF-a and IL-1. Clin Immunol. Immunopathol 50, 374. Roy, S., FITZ-GIBBON, L., POULIN, L. & WAINBERG, M.A. (1988) Infection of human monocytes/macrophages by HIV-1: effect on secretion of IL-1 activity. Immunology, 64, 233. VYAKARNAM, A., MCKEATING, J., MEAGER, A. & BEVERLEY, P.C. (1990) Tumour necrosis factors (a, Pi) induced by HIV- I in peripheral blood mononuclear cells potentiate virus replication. AIDS, 4, 21. WAHL, L.M., CORCORAN, M.L., PYLE, S.W., ARTHUR, L.O., HARELBELLAN, A. & FARRAR, W.L. (1989) Human immunodeficiency virus glycoprotein (gpl20) induction of monocyte arachidonic acid metabolites and interleukin 1. Proc. natl Acad. Sci. USA, 86, 621. WEISS, L., HAEFFNER-CAVAILLON, N., LAUDE, M., GILQUIN, J. & KAZATCHKINE, M.D. (1989) HIV infection is associated with the spontaneous production ofinterleukin-1 (IL-1) and with an abnormal release of IL-la in vitro. AIDS, 3, 695. WRIGHT, S.C., JEWETT, A., MITSUYASU, R. & BONAVIDA, B. (1988) Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients. J. Immunol. 141,99.