Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2769-2773, April 1993 Immunology The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes (AIDS/neurotoxin/brain/dementia/N-methyl-D-aspartate) DANA GIULIAN*t, ELAINE WENDT*, KEN VACA*, AND CHRISTINE A. NOONANO§ *Department of Neurology and tDivision of Molecular Virology, Baylor College of Medicine, Houston, TX 77030; and §Research Center for AIDS and HIV Infections, Veteran Affairs Medical Center, Houston, TX 77030 Communicated by Zanvil A. Cohn, January 8, 1993

ABSTRACT Mononuclear phagocytes infected with hu- METHODS man immunodeficiency virus 1 (HIV-1) produce soluble factors Cell Culture. Ciliary neurons were prepared from 9-day-old that kill neurons in culture. To defne the molecular events that chicken embryos, as described (8), and grown in 1.0 ml of N2 lead to neuron killing, HIV-1 proteins were tested for the ability medium/0.4% horse serum (BRL). After 2 days in culture, the to trigger release of neurotoxins from human monocytes and neurons were fixed and viewed at a magnification of X200 lymphocytes. None of the recombinant-derived HIV-1 proteins with phase-contrast microscopy. Neurons showing distinct examined (reverse transcriptase, protease, gag, nef, or gp120) nuclear membranes, characteristic nucleoli, and cytoplasms were directly neurotoxic at concentrations from 100 pM to 10 free of large vacuoles were scored as healthy cells. Neuron nM. The envelope glycoprotein gp120 did, however, stimulate killing scores, [(neurons per field of untreated control group both isolated human blood monocytes and the monocytoid line - neurons per field in treated group) x 100%]/neurons per THP-1 (but not lymphocytes or the lymphoid cell line 119) to field in untreated control group, are presented as mean values discharge neurotoxic factors. These toxins consisted of heat- + fields stable, protease-resistant molecules (<500 Da) that copurified SEM determined from at least 18 randomly selected with neurotoxins from HIV-1-infected THP-1 cells and were for each of, at least, three coverslips (8). The phase-contrast blocked by antagonists to N-methyl-D-aspartate receptors. scoring of neuron survival was confirmed by immunohisto- Release of neurotoxins through gpl20 stimulation involved chemical staining for choline acetyltransferase and neurofil- monocytoid CD4 receptors because toxin production could be ament (8) and by scanning EM (10). inhibited either by a monoclonal antibody to the CD4-binding To circumvent issues of species specificity, we also eval- region of gpl20 or by soluble CD4 receptors. Alternatively, uated neurons from spinal cord (day 14-15) and hippocampus production of neuron-killing factors could be induced with a (day 18) of rat embryos for neurotoxin sensitivity (6, 8, 11). peptide from the CD4-binding region of gpl20. These data Dissociated cells were grown in N2 medium/10% heat- show that the HIV-1 envelope glycoprotein alone can stimulate inactivated horse serum. Cultures were fixed after a 72-hr neurotoxin release by binding to CD4 receptors ofmononuclear exposure to control or conditioned medium. Neurons were phagocytes. Such neurotoxic factors may, in turn, contribute to identified in the rodent CNS cultures by indirect immuno- the central nervous system dysfunction associated with HIV-1 fluorescence by using a monoclonal antibody (mAb), MO3B, by acting on neurons through N-methyl-D-aspartate receptors. directed against the 160-kDa neurofilament protein (8). The number of neurons per field were scored as described above. Peripheral blood mononuclear cells were isolated from AIDS produces a devastating effect upon the brain and spinal buffy coats from HIV-1- and hepatitis B virus-seronegative cord with >70% of patients showing loss of memory, paral- individuals by density-gradient centrifugation on Ficoll/ ysis, seizures, sensory deficits, or global dementia (for re- Hypaque (12). The peripheral blood mononuclear cells were view, see ref. 1). Human immunodeficiency virus type 1 washed four times with phosphate-buffered saline (PBS) (pH (HIV-1) has been isolated from the central nervous system 7.4), resuspended in RPMI 1640 medium with L-glutamine (CNS) of AIDS patients and identified in several classes of (GIBCO)/10% fetal bovine serum/5% interleukin 2 (Cellular CNS mononuclear phagocytes-e.g., microglia, macro- Products), seeded into 100-mm plastic culture dishes at 20 x phages, and multinucleated macrophage-like cells (for re- 106 cells per 10 ml. After a 1-hr incubation at 37°C in 95% view, see ref. 2). Although direct viral infection of neurons in air/5% C02, nonadherent cells were removed from culture brain tissue has not been demonstrated by immunohistology dishes by gentle washing with three changes of warmed or by in situ hybridization (2), a recent ultrastructural study medium and replated at 2 x 106 cells per ml in plastic culture showed extensive neuronal damage (3). It has been proposed dishes for 24 hr. Flow cytometric analyses showed that >90% that AIDS-associated neurologic dysfunction stems from of the nonadherent cells contained the lymphocyte marker either the direct neurotoxicity of HIV proteins such as the CD45, whereas <1% were CD14(+) monocytes (13). Adher- envelope glycoprotein gp120 (4, 5) or some indirect mecha- ent cells were recovered by washes with ice-chilled Ca2+- and nism involving HIV-1-infected mononuclear phagocytes (6, Mg2+-free PBS and plated at 1 x 106 cells per ml. After a 24-hr 7). We favor the latter hypothesis because we have shown incubation, >95% of the adhering cells were monocytes, as that HIV-1-infected human monocytoid cell lines, but not an indicated by nonspecific esterase activity, scavenger low- infected human lymphoid line, produced a neurotoxic activ- density lipoprotein receptors, and the CD14 surface antigen ity (6) that functioned through the N-methyl-D-aspartate (13). Isolated lymphocytes or monocytes were maintained for (NMDA) type of glutamate receptor. We report here that 48 to 72 hr in RPMI 1640 medium or N2 medium containing gpl20, although not directly neurotoxic, can produce neu- 10% fetal bovine serum/5% interleukin 2. ronal damage by stimulating monocytes to release NMDA receptor-mediated toxins. Abbreviations: HIV-1, human immunodeficiency virus type 1; CNS, central nervous system; NMDA, N-methyl-D-aspartate; mAb, The publication costs of this article were defrayed in part by page charge monoclonal antibody; RP, reversed phase; APV, 2-amino-5- payment. This article must therefore be hereby marked "advertisement" phosphonovaleric acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2769 Downloaded by guest on September 26, 2021 2770 Immunology: Giulian et al. Proc. Natl. Acad. Sci. USA 90 (1993)

70 To monitor peptide binding to THP-1 cells, 10 ,ug of gpl20 60 _ gp120 peptide-(428-445) was radiolabeled in 20 ,ul of PBS (pH 7.2) 5o with 1 mCi of Na'25I (Iso-Tex, Arlington, TX; 1 Ci = 37 GBq) Y 40 [177] gag by the lodo-Gen (Pierce) method. After a 10-min reaction, the C 30 serum 0 mixture was added to 20 ,1 of bovine albumin at 1 L rt L 20 mg/ml with 10% PEG 1800 as carrier and passed over a 2-ml Z 10 z ..J] nef AG1-X6 (Bio-Rad) mini-column. The flow-through fraction 0 was concentrated on a Sep-Pak C18 cartridge (Waters), rinsed * -1 0 1 MRI4 ppro six times with 1 ml of PBS, six times with 1 ml of 10% -20 : L L In..l p m (vol/vol) acetonitrile, and eluted with 50% acetonitrile. The -30 eluate was dried in a vacuum concentrator (Speed-Vac, ciliary spinal hippocalmp Savant) and resuspended in PBS. Between 2-3 x 106 THP-1 FIG. 1. Recombinant HIV-1 proteins show no toxic effect upon cells per tube were incubated for 1 hr at 4°C with 120,000 dpm embryonic chicken ciliary neurons, embryonic rat spinal cord neu- of iodinated peptide in a total vol of 0.1 ml of RPMI 1640 rons, or embryonic rat hippocampal (hippocamp) neurons after 48 hr medium/bovine serum albumin at 2 mg/ml/10% fetal bovine of exposure at 10 nM concentrations. Observations are expressed as serum and then washed four times with 1 ml of medium. mean percentage of neuron kill SEMs with three coverslips per Percentage of specific binding was defined as [total dpm group from at least three independent experiments. Negative per- minus dpm in the presence of excess unlabeled gpl20 pep- centage neuron kill represents an increase in surviving neurons when tide-(428-445)]/total dpm. compared with controls. Pro, HIV-1 protease; rt, reverse transcrip- tase; gag, p25/24 gag. Recombinant HIV-1 Proteins and mAbs. Recombinant gpl20IIIB, gpl2OSF2, nef, p25/24 gag, reverse transcriptase, All established cell lines were cultured in RPMI 1640 and protease, were obtained from ARRRP. Recombinant medium with L-glutamine/10% fetal bovine serum, as de- HIV-1 proteins gp120IIB, reverse transcriptase, and syn- scribed above. The continuous human T-lymphocyte line thetic peptides gpl2O-(302-324) and gpl2O-(428-445) were (H9) was obtained through the AIDS Research and Reference purchased from American Biotechnologies (Cambridge, Reagent Program (ARRRP) (16) and the human monocyte cell MA). The anti-gpl20 mAbs, G3-42 and G3-213, map within to the V3 line THP-1 was from the American Type Culture Collection the CD4-binding site (amino acids 428-442) and (ATCC tumor immunology bank 202) (17). Derivation and loop, respectively, of gpl20 (20). characterization of HIV-1-infected cell lines have been de- RESULTS All lines used grew well under scribed (6). cell standard HIV-1 Proteins Are Not Neurotoxins. Picomolar concentra- culture conditions without any cytokines and were free of tions of gpl20, the major envelope glycoprotein of HIV-1, mycoplasma contamination, as determined by a ribosomal have been reported to be toxic to cultures of developing RNA-detection assay (Mycoplasma Rapid Detection Sys- neurons (4, 5, 21). To explore further the relationship be- tem, Gen-Probe, San Diego). tween HIV-1 proteins and neuronal injury, we tested the Biochemical Studies. Conditioned media were fractionated effects of various recombinant proteins on cultures of avian by ultrafiltration with YM-1 membranes (Amicon; estimated or rodent neurons. No direct killing was detected when molecular mass cut-off = 1000 Da) or by gel-filtration chro- chicken ciliary neurons, embryonic rat spinal cord, or rat matography (Bio-Gel-P2, Bio-Rad, 50.0 x 0.7 cm) using 50 hippocampal neurons were incubated with recombinant mM PBS as eluting buffer. Reversed-phase (RP) HPLC was HIV-1 proteins, including gpl20, p25/24 gag, nef, protease, done by using isocratic conditions (C18 Nova-Pak, 3.9 x 50 and reverse transcriptase, in concentrations from 100 pM to mm; Waters) with 10% acetonitrile/0.1% trifluoroacetic acid 10 nM (Fig. 1). These observations confirmed our previous (pH 2.2) at a flow rate of 1 ml per min. Amino acid concen- report (6) that neither HIV-1 proteins nor virions produced by trations were determined by a Waters PicoTag System. an HIV-infected lymphoid cell line killed neurons. Quantitative measurements of quinolinic acid levels in con- Mononuclear Phagocytes Release Neurotoxins in the Pres- ditioned medium (6, 8) were done with a Finnigan-MAT (San ence of gpl20. Because HIV-1-infected monocytoid cells can Jose, CA) Incos 50 gas chromatograph/mass spectrometer generate neurotoxic factor(s) (6), we next considered that and by a radioenzymatic assay (18). Levels of interleukin 1,3 HIV-1 proteins alone might stimulate human monocytes to and tumor necrosis factor a were measured by using ELISA release neurotoxins. Conditioned medium, collected after a kits (Quantakine, R & D Systems, Minneapolis), and throm- 48-hr exposure of the different cell types to various HIV-1 boxane B2 concentrations were determined by a RIA, as recombinant proteins, was tested for neurotoxic activity. At described by Hall and Tai (19). 10 nM concentrations, recombinant gpl20, but not p25/24 FIG. 2. Indirect toxicity of gp120 upon cili- A B ary neurons. Isolated human blood monocytes 60 60 (A) or human blood lymphocytes (B) were in- , cubated for 48 hr with 10 nM solutions of various 50 APV so APV recombinant-derived HIV-1 proteins. Media APV from the treated cells were then assayed at a Y 40 gEiZ .APV 40 final concentration of 20% (vol/vol) on ciliary 0 30 0 30 neurons for neurotoxicity. As shown, only , gp120 (gp)-stimulated monocytes released fac- 0 20 X 20 tors that were toxic to ciliary neurons (A). These 1 1 z effects were significantly elevated above un- aTTi* T iI treated control cultures (con) using Student's t 0 0 test (**, P < 0.001) with a confidence level of P -10 -10 < 0.006 by the Bonferroni method for eight con mono gp LPS zym nef gag rt pro con lymp gp LPS zym nef gag rt pro comparisons. Importantly, this toxic activity could be attenuated by the NMDA antagonist APV at 10 AM (A). Zymosan A particles (zym), but not lipopolysaccharide (LPS), also elicited neurotoxin release in the monocytoid cells (A) but did not elicit it in lymphoid cells (B). Media from unstimulated monocytes (mono) or lymphocytes (lymp) did not produce neuron-killing factors. Pro, HIV-1 protease; rt, reverse transcriptase; gag, p25/24 gag. Downloaded by guest on September 26, 2021 Immunology: Giulian et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2771

A B 0 Control 710T 70 o FIG. 3. Dose-response curves showing the neu- 6 60 ---gpl20/IB rotoxic activity released by monocytes (A) or 5o vT--v so THP-1 cells (B) exposed to 1 nM gpl20 from the C 4ko -L T -V- gp12O/SF IIIB or SF strains of HIV-1 (106 cells per ml _ 40 0 3 0 5 lo 1S 20.2530 incubated in N2 medium for 72 hr). Both forms of 10 30 -A- IIIB/APV gpl20 elicited the release of neurotoxic molecules. 2 z o 20 The action of the toxic molecules upon ciliary 10 0 -U- SF/APV neurons could be blocked by 10 ,uM of the NMDA 0 antagonist APV. A similar dose-response is shown -1 -10 --- THP/HIV for medium conditioned by HIV-1-infected THP-1 0 5 10 15 20 25 30 0 5 10 15 20 25 30 cells (B). Controls represent media from cells pre- pared in an identical fashion but without HIV-1 % Volume % Volume proteins. Data represent mean values + SEMs.

gag, reverse transcriptase, nef, or protease, stimulated the CD4 Receptors Mediate Production of Neurotoxins. Infec- release of neurotoxicity from both isolated human blood tion of macrophages and microglia by HIV-1 is mediated monocytes (Fig. 2A) and the monocytoid cell line THP-1 through the CD4 receptor, a gpl20-binding site (26, 27). To (data not shown). Importantly, the action of the neurotoxin explore the role of the CD4 receptor in gp120-induced pro- could be blocked by the selective NMDA-receptor antagonist duction of neurotoxins, recombinant soluble CD4 (28) was 2-amino-5-phosphonovaleric acid (APV) (Fig. 2A) (22). A added to THP-1 cultures before incubation with 1 nM gpl20. similar blockade has been described for neurotoxins from Under these conditions, no neurotoxin production was de- HIV-1-infected cells (6). No toxic activity was obtained from tected in the presence of soluble CD4 at >10,ug/ml (Fig. 4A), human blood lymphocytes (Fig. 2B) or from H9 lymphoid an amount ofsoluble CD4 equivalent to that required to block cells (data not shown) after treatment with gpl20 or any ofthe HIV-1 infection of lymphocytes (28). Moreover, the mAb, other HIV-1 proteins. G3-42, which maps to a region within the CD4-binding site of Dose-response curves for medium conditioned by blood gpl20 (20), could suppress the gpl20 stimulation of neuro- monocytes (Fig. 3A) or the THP-1 cell line (Fig. 3B) exposed toxin release at .3 ,ug/ml (Fig. 4A) or that concentration of to 1 nM gpl20 showed that the neurotoxin production re- antibody needed to block HIV-1 infection of lymphocytes sembled that of HIV-1-infected THP-1 cells (Fig. 3B). A (20). In contrast, a second mAb (G3-213), which maps to the recombinant gpl20 derived from the SF2 strain (gpl2OSF2) of V3 loop of gpl20, did not alter gp120-induced production of HIV-1 also stimulated release of neurotoxicity (Fig. 3 A and neurotoxic activity (Fig. 4A). B), suggesting that the activation of mononuclear phagocytes To confirm that the CD4-binding region of gp120 was was not HIV-1 strain specific. Moreover, gp120-elicited necessary to elicit the production of neurotoxins, the effects neurotoxins killed neurofilament(+) spinal cord neurons of of different gpl2OhIIB-derived peptides upon neurotoxin se- embryonic rat (data not shown), which argues against a cretion by THP-1 cells were evaluated. The 1251-labeled species-restricted mechanism of killing. As noted for ciliary peptide-(428-445), which included the CD4-binding region neurons, spinal neurons were protected from monocyte- (29), showed -50% specific binding to THP-1 cells, and derived neurotoxin(s) by APV. >90o of this specific binding was blocked by 10 nM recom- To establish whether production of neurotoxins by mono- binant gpl20(HIVIIIB) or gpl20(HIVsF2) but was not blocked nuclear phagocytes was a process specific to HIV-1 infection, by 10 nM of the V3 loop (30) peptide-(302-340). Peptide- we examined the effects of other immuno-stimulants upon (428-445) elicited neurotoxic activity from THP-1 cells, mononuclear cells. Yeast wall particles, zymosan A, also whereas the V3 loop peptide-(320-345) did not (Fig. 4B). caused release of neurotoxins from both blood monocytes Moreover, mAb G3-42, which binds to overlapping residues (Fig. 2A) and THP-1 cells, but did not cause release from 428-440, blocked the effects of peptide-(428-445) (Fig. 4B). blood lymphocytes (Fig. 2B) or H9 cells. Importantly, no Thus, gp120 stimulated the release of neurotoxins by binding neurotoxins were produced by monocytes or lymphocytes to the CD4 receptors of mononuclear phagocytes. exposed to lipopolysaccharide (Fig. 2), ruling out endotoxin Characterization of gp12O-Induced Neurotoxic Activity. Ul- contamination as a confounding factor (23). Although zymo- trafiltration analyses suggested the neurotoxic activity con- san A and lipopolysaccharide stimulated release of throm- sisted of small molecules (<1 kDa). Further analysis by boxane B2 (24) and interleukin 13 (25) (Table 1), only zymo- gel-filtration chromatography indicated that the activity had a san A and gpl20 were able to elicit the production of molecular mass of <500 Da (data not shown). This cytotoxic neurotoxins. Therefore, activated mononuclear phagocytes activity was heat-stable and protease-resistant whether recov- clearly could generate neurotoxins without HIV-1 infection ered from gpl2O-stimulated normal blood monocytes, gpl20- or exposure to individual HIV-1 proteins. stimulated THP-1 cells, or from HIV-1-infected THP-1 cells. Table 1. Monocyte activators and secretion products Neurotoxicity, TNF-a, pg/ml IL-1/3, pg/ml TxB2, ng/ml % kill Control 53.2 ± 1.2 (4) 70.5 ± 3.8 (4) 6.7 ± 0.1 (4) 0 ± 6 (5) gpl20 49.8 ± 0.3 (4) 61.0 ± 6.8 (4) 5.7 ± 0.7 (4) 45 ± 4 (4) Zymosan A 1258.8 ± 9.3 (4) 1962.0 ± 158.0 (3) 131.2 ± 4.0 (4) 42 ± 3 (4) LPS 514.6 ± 7.1 (4) 292.0 ± 47.0 (4) 142.9 ± 3.2 (4) 5 + 2 (4) Isolated human blood monocytes (106) were incubated for 72 hr in the presence of N2 medium containing either 1 nM recombinant gpl20, suspended zymosan A particles (320 Ag/ml), or lipopolysaccharide (LPS) at 10 /g/ml. Neurotoxic activity was determined by using the ciliary neurons in the presence of20% (vol/vol) conditioned medium. As shown, gp120, but not LPS, stimulates monocyte release of neurotoxic activity. In contrast, LPS, but not gpl20, stimulates release of the cytokines tumor necrosis factor a (TNF-a) and interleukin 1p3 (IL-1,8). These data suggest that different mechanisms regulate cytokine and neurotoxin production. Numbers in parentheses are the numbers of stimulated cultures tested. TxB2, thromboxane B2. Downloaded by guest on September 26, 2021 2772 Immunology: Giulian et al. Proc. Natl. Acad. Sci. USA 90 (1993) A B 60 70 -0- 428-445

T 60 T T -*- G3-42 50 **. *- 302-324 0 L~~~~~~~~~~~~~~- 40 40 0 I I C C 0 --°-- G3-213 ° 30 -0- 428/G3-42 '- 30 m 20 4) 4) Z -v- sCD4 Z TT --- 428/G3-213 20 ~~~~~~~T lo i 8 O0 ....1 10 0 i -10 -v- 428/APV 0 I -20 lo-, 1 00 10' 10' 0 5 10 15 20 25 30 Protein (pg/ml) % Volume FIG. 4. Activation of THP-1 cells by gp120IIIB depends upon the CD4 receptor. (A) A recombinant form of the soluble CD4 receptor (sCD4) prevented the ability of gpl20 (48-hr exposure at 1 nM) to elicit neurotoxin production. Near-total blockade occurred when SCD4 was at >2 Ag/ml, which corresponded to the concentration required to prevent HIV-1 infection of lymphocytes. The mAb G3-42, which recognized a CD4-binding epitope on gpl20, prevented gp120-elicited release of neurotoxic activity. In contrast, mAb G3-213, which recognized the V3 loop region of gp120, did not block stimulated production of neurotoxins. (B) Peptides derived from gpl20 elicited neurotoxin production in THP-1 cells. Cells were incubated with 1 nM of synthetic peptide-(428-445), which includes the CD4-binding site, or with 1 nM of peptide-(302-324) from the V3 loop. As shown, only that portion of the gp120 molecule that corresponded to the CD4-binding region elicited toxin production. Although mAb G3-42 bound to peptide-(428-445) and blocked its stimulatory action (428/G3-42), control antibody G3-214 was unable to block the stimulation (428/G3-213).

Dose-response curves showed that toxin concentrations that nor the excitotoxin quinolinic acid coeluted with the toxic killed neurons had no apparent effect on cultured Schwann activity (Fig. 6A). Moreover, amino acid analyses of the cells, astroglia, oligodendroglia, or microglia (data not shown). toxin-containing fractions showed that the levels of excit- Use of receptor antagonists and channel blockers demon- atory amino acids (glutamate < 2 nM; aspartate < 2 nM) were strated that only the NMDA class of glutamate receptors below the concentrations normally found in culture medium mediated the actions of gpl20-associated toxins upon either (6, 8) and, therefore, could not account for the observed ciliary or spinal neurons (Fig. 5). In contrast, two antagonists neuron-killing effects (data not shown). Similarly, the amount to non-NMDA-type excitatory amino acid receptors (6-cyano- 7-nitroquinoxoline-2,3-dione and y-D-glutamylaminomethyl- A sulfonic acid, ref. 31) had no protective effect (Fig. 5). 70 Neurotoxins that involve NMDA-receptor mechanisms 0 60 C include excitatory amino acids and their metabolites (31, 32). 50 -0- gpl20 To compare the HIV-1-associated neurotoxins with such Y 40 C --°-- control known toxins as glutamate and quinolinic acid (33, 34) a 0 30 of the was done partial purification toxic activity by using 3 20 ultrafiltration followed by RP-HPLC. Neurotoxic activities -A - APV eluted from RP-HPLC in identical patterns whether obtained 10 from THP-1 cells stimulated by gpl20 (Fig. 6A) or infected - 1 0 with HIV-1 (Fig. 6B). These toxic activities were blocked by -10' APV and could not be detected in control cells. Importantly, 0 2 4 6 8 10 12 14 neither the excitatory amino acids, glutamate and aspartate, B 70 ~~~~120~~gp 60 60 ._50 -v- HIV-1

+~L APV c 40 0 - 0- control 4 0 '- 30 ~~~~~~~MK801 20 z -A - APV 10

0 1 0

+GAMS -10 0~ ~~~ 0 2 4 6 8 10 12 14 -10 L I-rr-- ciliary spinal Fractions

FIG. 5. Protective effects of drugs after exposure to neurotoxic FIG. 6. Fractionation of neurotoxic activity from monocytes or activity released by THP-1 cells stimulated with 1 nM gpl2OIIIB. Both from THP-1 cells by RP-HPLC. After a 48-hr incubation, the condi- ciliary neurons and spinal neurons showed enhanced survival in the tioned media were recovered as an ultrafiltrate (<1 kDa), eluted on a C18 presence of the NMDA glutamate-receptor antagonist APV (10 ,uM) column (10% acetonitrile/0.1% trifluoroacetic acid at 1 ml per min) and or the NMDA-mediated channel blocker MK-801 (10 ,uM). In con- recovered in 1-ml fractions. (A) A peak of neurotoxic activity appeared trast, the non-NMDA glutamate-receptor antagonists, 6-cyano-7- in gpl20InIB-stimulated monocytes (gp120) but not in unstimulated cells nitroquinoxoline-2,3-dione (CNQX) and y-D-glutamylaminomethyl- (control). This toxic activity was blocked by the NMDA antagonist sulfonic acid (GAMS), were not protective. Data are expressed as APV. (B) An identical profile was noted for the neurotoxic activity mean values SEMs. These effects were significantly elevated released by HIV-1-infected THP-1 cells (HIV-1). The monocyte- above the untreated control cultures (controls) using Student's t test derived or THP-1-derived neurotoxins could be separated from quin- (*, P < 0.001) with a confidence level of P < 0.01 by the Bonferroni olinic acid (qa), aspartic acid (asp), and glutamic acid (glu). Values are method for five comparisons. expressed as mean percentage neuron killing ± SEMs. Downloaded by guest on September 26, 2021 Immunology: Giulian et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2773 of quinolinic acid detected in those fractions from RP-HPLC neuropathology and could cause the loss of cortical neurons that had neurotoxic activity was far below an effective toxic seen in HIV-1-infected individuals (6, 39). concentration, as determined by gas chromatography/mass We thank Dr. Dorothy Lewis, Baylor College ofMedicine, forflow spectrometry or by enzymic assay (<5 nM) (6, 8). Unlike cytometry analysis, Dr. Clay Goodman, Baylor College ofMedicine, excitatory amino acids, the gp120-elicited neurotoxic activity for amino acid analyses, Dr. Shen-Nan Lin, University of Texas did not bind to strong anionic exchangers-e.g., Dowex-l- Health Science Center at Houston, for gas chromatograph/mass and lacked carboxylic acid moieties, as determined by buta- spectrometric analyses, and Dr. Robert Schwarcz, University of nol esterification in concentrated HCI. In contrast to such Maryland, for radioenzymatic determinations ofquinolinic acid. We also thank Dr. Tse-Wen Chang ofTanox Biosystems, Inc. (Houston) glycerolipids as platelet-activating factor, the neurotoxin for two anti-gp120 mAbs and Dr. Arlene Chiu (Beckman Research could not be isolated by lipid-extracting solvents (at pH 7.0) Institute, City of Hope, CA) for the anti-neurofilament antibody and resisted acid hydrolysis (24 hr at 105°C). MO3B. This work was supported, in part, by Grant RO1 MH48652 and Grant RO1 NS23115 from National Institute of Mental Health, DISCUSSION by Grant 500056 from the American Foundation for AIDS Research, Mononuclear phagocytes (macrophages, microglia, and mul- by an American Foundation for AIDS Research Student Fellowship tinucleated giant cells) are the principal cellular targets for to E.W., and by the Veteran Affairs Medical Center, Houston. HIV replication in the CNS. Because HIV-1 infection of 1. Navia, B. A., Jordan, B. D. & Price, R. W. (1986) Ann. Neurol. 19, 517-524. neurons occurs rarely, if at all, the mechanisms involved in 2. Price, R. W., Sidtis, J. J., Navia, B. A., Pumarola-Sune, T. & Ornitz, the induction of neurologic impairment in HIV-1-infected D. B. (1988) in AIDS and the Nervous System, eds. Rosenblum, M. L., patients remain obscure. Proposed etiologies have included Levy, R. M. & Bredesen, D. E. (Raven, New York), pp. 203-219. 3. Wiley, C. A., Masliah, E., Lenere, M., Morey, M., Lemere, C., DeTeresa, direct neuron killing by the HIV-encoded proteins, gpl20 (4, R., Grafe, M., Hansen, L. & Terry, R. (1991) Ann. Neurol. 29, 651-655. 5) and tat (35), and by soluble factors produced by HIV- 4. Brenneman, D. E., Westbrook, G. L., Fitzgerald, S. F., Ennist, D. L., infected monocytoid cells (6, 7). Elkins, K. L., Ruff, M. R. & Pert, C. B. (1988) Nature (London) 335, 639-642. In contrast to Brenneman et al. (4) and Lipton and co- 5. Dreyer, E. B., Kaiser, P. K., Offermann, J. T. & Lipton, S. A. (1990) workers (5, 21), we found no evidence for direct neuron Science 248, 364-367. killing by gpl20 in three different neurotoxin assays involving 6. Giulian, D., Vaca, K. & Noonan, C. A. (1990) Science 250, 1593-1595. avian 7. Pulliam, L., Herndier, B. G., Tang, N. M. & McGrath, M. S. (1991) J. and rodent cells. We do show, however, that gpl20 Clin. Invest. 87, 503-512. binding to the CD4 receptor caused human monocytoid cells 8. Giulian, D., Vaca, K. & Corpuz, M. (1993) J. Neuroscience 13, 29-37. to release neurotoxic activity. This toxicity, in turn, killed 9. Stone, T. & Connick, J. (1985) Neuroscience 15, 597-617. avian and rodent neurons 10. Giulian, D. (1993) Glia 7, 102-110. in a manner dependent upon 11. Banker, G. A. & Cowan, W. M. (1977) Brain Res. 126, 397-425. NMDA receptors. The neurotoxic substances from HIV- 12. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89. infected- or gp120-stimulated monocytoid cell release can be 13. Lewis, D. E. & Richman, W. J. (1992) Manual of Clinical Laboratory distinguished from other known classes of monocyte- Immunology, eds. Rose, N. R., de Marcio, E. C., Fahey, J. L., Fried- man, H. & Penn, G. M. (Am. Soc. Microbiol., Washington), 4th Ed., pp. associated cytotoxins (36-38) but show similar physical 164-173. properties to neurotoxins recovered from rat microglia and 14. Heyes, M. P., Rubinow, D., Lane, C. & Markey, S. P. (1989) Ann. macrophages (8, 10, 36). Neurol. 26, 275-277. 15. Piani, D., Frei, K., Do, K. Q., Cuenod, M. & Fontana, A. (1991) There are several plausible candidates for HIV-1 neuro- Neurosci. Lett. 133, 159-162. toxins (39). As with the HIV-1-associated neurotoxins de- 16. Popovic, M., Samgadhaaran, M. G., Read, E. & Gallo, R. C. (1984) scribed here, quinolinic acid is a small, heat-stable neuron Science 224, 497-500. 17. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T. poison that acts through NMDA receptors (9). Moreover, & Tada, K. (1980) Int. J. Cancer 26, 171-174. Heyes et al. (14) reported marked elevations in quinolinic acid 18. Foster, A. C., Okuno, E., Brougher, D. S. & Schwarcz, R. (1986) Anal. levels in the cerebrospinal fluid of patients suffering from Biochem. 158, 98-103. 19. Hall, E. R. & Tai, H.-H. (1985) Biochim. Biophys. Acta 665, 498-503. AIDS dementia or a number of other disorders including 20. Sun, N.-C., Ho, D. D., Sun, C. R. Y., Liou, R.-S., Gordon, W., Fung, hepatic coma, meningitis, CNS neoplasm, stroke, and CNS M. S. C., Li, X.-L., Ting, R. C., Lee, T.-H., Chang, N. T. & Chang, parasitic infections. However, we did not find that HIV-1- T. W. (1989) J. Virol. 63, 3579-3585. infected cells or monocytes stimulated with gpl20 released 21. Kaiser, P. K., Offermann, J. T. & Lipton, S. A. (1990) Neurology 40, 1757-1761. cytotoxic levels of quinolinic acid. Moreover, quinolinic acid 22. Dingledine, R. (1986) Trends Neurosci. 9, 47-49. did not copurify with the neurotoxins reported here. Recently, 23. Nathan, C., Murray, H. & Cohn, Z. (1980) N. Engl. J. Med. 303, 622-625. Piani et al. (15) have reported that cultured microglia consti- 24. Bonney, R. J. & Humes, J. L. (1984) J. Leukocyte Biol. 35, 1-10. tutively release levels of glutamate that under some in vitro 25. Molina, J.-M., Scadden, D. T., Amirault, C., Woon, A. & Vannier, E. (1990) J. Virol. 64, 2901-2906. conditions are toxic to neurons. We did not measure signifi- 26. Dalgleish, A. G., Beverley, P. C. L., Clapham, P. R., Crawford, D. H., cant release of glutamate by activated mononuclear cells. Greaves, M. F. & Weiss, R. A. (1984) Nature (London) 312, 763-767. More importantly, glutamate was not toxic to ciliary neurons 27. Jordan, C. A., Watkins, B. A., Kufta, C. & Dubois-Dalcq, M. (1991) J. (8, 10) nor did it copurify with the neurotoxins described here. Virol. 656, 736-742. Although we have to 28. Deen, K. C., McDougal, J. S., Inacker, G., Folena-Wasserman, G., yet identify the long-lived neurotoxic Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R. & Sweet, R. W. (1988) factors produced by HIV-1-infected cells, these molecules can Nature (London) 331, 82-84. be distinguished by molecular mass and stability, RP chroma- 29. Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shimasaki, C., tography, and biologic actions from a number of substances Patzer, E., Berman, P., Gregory, T. & Capon, D. J. (1987) Cell 50, 975-980. described either as neuron-killing factors endogenous to the 30. Hwang, S. S., Boyle, T. J., Lyerly, H. K. & Cullen, B. R. (1991) Science brain or as cell poisons secreted by macrophages (10). 253, 71-73. Although in vitro experiments involving recombinant pro- 31. Honore, T. (1989) Med. Res. Rev. 9, 1-23. teins or synthetic peptides might not reflect actions of native 32. Roberts, P. J. & Davies, S. W. (1987) Biochem. Soc. Trans. 15, 218-219. 33. Meldrum, B. (1985) Trends NeuroSci. 8, 47-48. gp120 under physiologic conditions, we conclude that the 34. Schwarcz, R., Whetsell, W. 0. & Mangano, R. M. (1983) Science 219, envelope glycoprotein gpl20 alone was sufficient to elicit 316-318. neurotoxin production in cultured human mononuclear 35. Sabatier, J.-M., Vives, E., Mabrouk, K., Benjouad, A., Rochat, H., phagocytes. Moreover, this induction process involved bind- Duval, A., Hue, B. & Bahraoui, E. (1991) J. Virol. 65, 961-966. ing to CD4 receptors. We believe 36. Giulian, D. (1992) Curr. Top. Neurol. 12, 23-54. that this secretion of 37. Giulian, D. & Baker, T. J. (1986) J. Neurosci. 6, 2163-2178. neurotoxic factors by HIV-1-infected mononuclear phago- 38. Thery, C., Chamak, B. & Mallat, M. (1991)Eur. J. Neurosci. 3,1155-1164. cytes represents a basic mechanism for HIV-1-associated 39. Giulian, D. & Noonan, C. (1992) AIDS Res. Rev. 2, 157-170. Downloaded by guest on September 26, 2021