Res. Immunol. 1992, 143, 49-56
Functional R. LeNaour(
consequences of monocyte/macrophage infection by HIV1 H. Raoul(‘), A. Mabondzo (l), L. Ripoll(‘), J. Bartholeynst2), J.L. Romet-Lemonne(3) and D. Dormont(‘14)
(“Laboratoire de Neuropathologie expkrimentale et Neurovirologie, Commissariat ci I’Energie Atomique, CRSSA/DS V/DPTE, B. P. 6, 92265 Fontenay-aux-Roses Cedex (France), Q) CNTS, B.P. 100, 91943 Les Ulis Cedex (France) and 13’1. D.M., 91930 Orsay Cedex (France), and (4’Institut Pasteur, Paris SUMMARY
Monocyte/macrophage infection by human immunodeficiency virus type 1 (HIV1 1was studied for its effects on the production of tumour necrosis factor a (TNFa) and the expression of the manganese superoxide dismutase (MnSOD) gene. For this purpose, human peripheral blood monocytes were obtained from healthy HIV1 -seronegative donors by centrifugal elutriation and infected with either the HIVIILAVI strain or with the primary HlVl/DAS isolate. The results showed that (I I HIVl/LAVl-infected macrophages did not produce any biologically detectable TNFa during the few hours following lentiviral infection, despite rises in the TNFa mRNA level; (21 MnSOD gene transcription in the macrophages increased, as measured 2 and 4 h after infection; (3) the level of the MnSOD gene expression declined during the late phases of lentiviral infection, but TNFa synthesis and gene expression rose; and (4) bispecific antibody comprised of antiFcyRl (anti-CD641 and anti-gp41 monoclonal antibodies inhibited the in vitro infection of monocyte-derived macrophages by HIV 1 IDAS. Key-words: HIV, Macrophage, TNFa, Superoxide dismutase; Microbicidal activity, Viral replication, MnSOD gene, Bispecific antibody.
Human monocytes/macrophages (HMM) are major participants in the host defence against numerous pathogenic microorganisms. When HMM are infected by the human immunodeficiency virus (HIV) -the aetiologic agent of acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983) they serve as viral targets and viral reservoirs (Ho et al., 1986; Gartner et al., 1986; McElrath et al., 1989, 1991). During this infection, HMM also act as regulators that seem to control viral replication and the progression of disease by dysregulation of their own cytokine synthesis and microbicidal activity. Several authors have reported that HIV long terminal repeat(LTR)-directed gene expression increases in differentiated monocytes after exposure of the latter to various cytokines including monocyte colony-
stimulating factor, granulocyte-macrophage colonystimulating factor, interleukin-1 (ILl), IL4 and tumour necrosis factor a (TNFa). In particular, TNFa enhances the replication of HIV1 (Matsuyama et al., 1989), stimulates HIV1 gene expression (Okamoto et al., 1989) and induces a nuclear factor that binds to the nuclear factor xB sites on the HIV LTR of T cells and macrophages (Osborn et al., 1989; Duh et al., 1989). Furthermore, elevated levels of TNFa were measured by double antibody radioimmunoassay in sera from individuals with AIDS (Lahdevirta et al., 1988). Secretory products released from macrophages, including H,O,, 0 ; , IL 1 and TNFa serve as effector molecules in microbicida activity, and thus the amount of these products increases after in vitro treatment with interferon-y of monocytes recovered from HIV1 infected individuals (Murray et al., 1984, 1987, 1988). Likewise, monocytes from such in-
dividuals display an oxidative burst and microbicida1 activity towards Toxoplumta gondii and Chlamydia psittaci, which behave like intramacrophagic parasites. To grasp fondamental functions of HMM in the pathogenesis of lentiviral infection, a better understanding of the way in which HMM are infected is necessary. Several observations suggest the interaction of HIV gpl20 with CD4 molecules expressed at the macrophage surface, but this does not preclude other pathways of infection. For instance, HIV may enter macrophages through FcR-independent and/ordependent phagocytosis (Takeda et al., 1988 ; Homsy et al., 1989). Within this framework, it was of interest to study in vitro (1) the consequences of HIV infection of monocytes/macrophages for the regulation of TNFcr synthesis; (2) the expression of the manganese superoxide dismutase (MnSOD) gene, as this enzyme protects cells from the toxicity of O,, and (3) the role of the CD4 and FcyRI molecules m the mechanisms controlling the entry of HIV1 into macrophages. Materials
Human peripheral blood monocytes were obtained from healthy HIVl-seronegative donors using the centrifugal elutriation technique (Beckman J2-21/ME centrifuge, JE-B6 rotor). Freshly isolated monocles were analysed by flow cytometry with a fluorescence-activated cell sorter analyser (FACS) (Becton-Dickinson). Cells were more than 95% positive for CDllb (a-chain of the CR3,C3 bireceptor) and less than 1% positive for CD4 and CD8 After isolation, cells were cultured on microtitre plates or plastic flasks in RPMI-1~0 medium supplemented with 10% heat-inactivated fetal calf serum (Boehringer Mannheim) ; 100 ug penicillin/streptomycin/neomycin and 2 mM Lglutamine. Seven days after isolation, adherent cells were scraped off the supports and cell surface antigens were re-analysed for the same surface molecules using the same monoclonal antibodies (mAb) and for the Max1 antigen (anti-Max1 mAb was kindly provided by Dr Andreesen, Fribourg, Germany). The latter antibody recognizes the differentiation antigen
HMM IL L-I-R mAb MDM
= = = = =
human monocyte/macrophage. interleukin. fong terminal repeat. monoclonal antibody. monocyte-derived macrophage.
ET AL. Maxi, markedly increased after in vitro differentiation (Andreesen et al., 1986). Cells were 95% CDllb+, 48% Maxl’, and CD4- and CD88. Viral strains Seven-day old monocyte-derived macrophages (MDM) were infected either with the HIVl/LAVl strain or with the primary isolate HIVl/DAS, isolated from a patient who had developed acute regressive encephalopathy (Boussin et al., 1987). HIVl/LAVl and HIVl/DAS stocks had respective infectivity titres of lo5 and 10’ tissue culture infective doses 50% (TCID&/ml, as measured by endpoint microtitration of HIV in a 96-well microplate assay using MT2 cells (Dougherty et al., 1964).
Bispecific antibody was obtained by the linkage of anti-HIV glycoprotein gp41 and anti-FcyRI (antiCD64 mAb) Fab’ fragments. The human anti-gp41 mAb recognizes an epitope localized in the junction zone of gp120-gp41, whereas the murine anti-FcyRI mAb recognizes the type I receptor for the Fc fragment of immunoglobulins expressed at the surface of human mononuclear phagocytes (Anderson et al., 1986). Bispecific antibodies were synthesized by pepsin digestion of each whole monoclonal antibody. The two F(ab’), fragments thus obtained were treated by a disulphide bond reduction, and covalent binding of the two resulting Fab’ fragments produced a bispecific antibody. HIVZ infection
I) HIVl/LA Vl infection. - Seven days after isolation, MDM were washed three times in phosphatebuffered saline 1 x and were infected with 0.02 TCID,,/cell of HIVl/LAVl. This infection occurred in 7 ml of culture medium supplemented with 10% foetal calf serum, Two to 10 h after infection, culture supernatants were recovered for a TNFa cytotoxicity assay, and cells were washed, lysed and their RNA extracted for Northern blot analysis. In long-term-infected macrophage cultures, MDM were washed 24 h after infection and cultured in fresh RPMI-1640 medium supplemented with 10% fcetal
MnSOD RT TCID, TNFu
= = = =
manganese-superoxide dismutase. reverse transcriptase. 50% tissue culture infective dose. tumour necrosis factor a.
calf serum. Infected MDM culture supernatants were recovered every 2 days for TNFu assay, and viral replication was measured by reverse transcriptase (RT) activity. 2) HIVI/DAS infection. - The bispecific antibody Fab’ anti-gp41/FcyRI was serially diluted from a stock solution at 152 Kg/ml ; 100 ~1 of each dilution were preincubated for 30 min at 37°C with 100 ~1 of HIVl/DAS (10,000 cpm of RT activity per ml). Immunecomplexes were then added either directly to the MDM cultures or to cultured pretreated with anti-Leu3a MDM at a final concentration of 2.5 pg/ml. Twenty four hours after infection, cells were extensively washed and cultured in RPMI-1640 medium with 10% fetal calf serum; for the pretreated MDM cultures, the same fresh medium was used after their infection, supplemented with anti-Leu3a throughout culture (final concentration; 2.5 pg/ml). Viral infection was monitored every 4 days by measuring the amount of P24 antigen in culture supernatants of infected MDM. Virus replication
The RT activity of the HIV1 in the cell culture supernatants was measured as previously described (Rey et al., 1984). Briefly, the supernatants were ultracentrifuged for 5 min at 10,000 rpm (Beckmann TLlOO) and viral pellets were resuspended in 20 ~1 of NTE (10 mM NaCl ; 10 mM Tris ; 1 mM EDTA) containing 0.1 per cent Triton-X100. Next, 10 IJ-I of viral lysate were added to a reaction mixture containing 5 mM MgCI,, 1 mM dithiothreitol, 2.5 Kg/ml as template-primer, and poly(rA)-oligo(dT)l,~ls 0.2 nmol of methyl-‘H-thymidine triphosphate (Amersham Corp., TRK 354, 1.1 TBq/mmol). The mixture was then incubated for 1 h at 37”C, placed on nitrocellulose filters and extensively washed. Filters were dried and RT activity was quantified by measuring the incorporated radioactivity. The P24 antigen level in culture supernatants was determined by an ELISA (enzyme-linked immunosorbent assay) method (P24 antigen capture Abbott kit). Bioassay for TNFu activity Culture supernatants were assayed for TNFct by a cytotoxicity assay using L-929 cells as described by Fish and Gifford (1983). Briefly, cells of the murine L-929 cell line were recovered after trypsinization during the exponential growth phase, washed several times, seeded in 96-well microtitre plates at a density of 5 x lo4 cells per well, and incubated for 2 h at 37°C. Next, 100 ~1 of each infected and uninfected culture supernatants was placed in each well with the adherent L-929 cells. Twenty ~1 of actinomycin D
(final concentration: 1 pg/ml) was then added to each well, and the plates were incubated for 24 h. Viable cells were stained with crystal violet, and cytotoxicity was quantified with a microtitre plate reader (wavelength : 540 nm). Results were expressed in picograms per millilitre of TNFa, according to a standard curve constructed on the basis of serial dilutions of recombinant human TNFu. RNA extraction and Northern
After different periods of culture, infected or noninfected MDM were scraped off their supports and lysed with guanidine isothiocyanate. RNA were then prepared as described by Chomczynski et al. (1987). Briefly, cytoplasmic RNA were extracted by the phenol-chloroform method, precipitated in isopropanol at -20°C for 2 h and washed twice in 75% ethanol. The RNA preparations were then resuspended in a solution of 5 mM EDTA and 0.1% diethylpyrocarbonate. RNA concentrations were determined by measuring absorbance at 260 nm. Northern blot analysis was performed as follows : RNA were separated by electrophoresis through a formaldehyde 1.5% agarose gel, transferred overnight onto a nylon filter (Schleicher and Schuell) by capillarity with 20 x SSC buffer, and fixed by UV exposure. Filters were hybridized overnight at 42°C in the presence of 50% formamide, washed and autoradiographied at - 80°C. Probes Three probes were used: (1) a p-actin nicktranslated cDNA probe (Dr. S. Alonso, Paris, France) for internal experimental control (Alonso et al., 1986), (2) a TNFa probe consisting of a cDNA clone of human TNF in PAT153 (Prof. Fiers, Ghent, Belgium), and (3) an MnSOD probe consisting of a cDNA clone of human MnSOD in pSP65 (Ph. D, thesis; Ye Shih Ho, Durham, USA).
TNFu synthesis in HIVI-infected natants
TNFu synthesis in the supernatants of uninfected or HIV1 /LAVl-infected MDM was assayed every 2 h for 10 h. TNFa was below the positivity threshold of 36 pg/ml for recombinant human TNFa, both in uninfected and infected cultures (fig. 1). After 2 and then 4 h of infection, TNFu mRNA were detected by Northern blot analysis only in infected cultures (fig. 2A). In contrast, no specific mRNA were detected at the same time-points with a human
=1 B 5 I
Time (hours) Fig. 1. TNFa synthesis in the supematants of HIVl-infected
MDM during the first ten hours of infection. (0) = TNFa synthesisin infected macrophageculture supernatants,(0) = TNFa synthesis in uninfected culture supernatants.Cut off isexpressed asthe meannegativecontrol -+ 2 SEM.
IL6 cDNA probe in either infected or uninfected cultures (data not shown). However, when HIV 1-infected MDM cultures were at the time of high viral replication, maximal TNFa synthesis was observed several days after infection with HIV1 at the time of high viral replication : at day 13 after infection (20 days post-isolation), 365 pg/ml of TNFu were detectable in MDM-infected culture compared to uninfected culture, and a peak of RT activity was measured (table I). Contrary to uninfected cultures, TNFa mRNA synthesis in infected cultures rose between 3 and 13 days post-infection. At day 15 post-infection, no significant TNFa mRNA signal was observed in either type of culture (table I). In infected MDM cultures, no TNFa. synthesis was detected even when RT activity was negative and P24 antigen positive. In all experiments, signal intensity was normalized by densitometric analysis using a constitutively expressed p-actin probe. MnSOD gene expression
To determine whether HIV1 induced MnSOD gene transcription through a mechanism associated with viral entry and host cell membrane stimulation, RNA were analysed during the hours following MDM infection. At 2 and 4 h, increases were observed in the level of MnSOD gene expression,
Time (hours) Fig. 2. TNFa andMnSOD geneexpressionduringthe early
eventsof HIV1 macrophageinfection. The RNA isolatedfrom uninfectedand infectedmacrophages were measured by densitometric analysis of Northern blot (signal intensity was normalized using a constitutivelyexpressed p-actinprobe. Resultsareexpressed in arbitrary units. A) TNFa gene expressionin HIVl-infected human MDM (grey column) and uninfected human MDM (hatchedcolumn). B) MnSOD geneexpressionin HP/l-infected human MDM (grey column) and uninfected human MDM (hatchedcolumn).
compared to the negative control (fig. 2B). Six hours after infection, this level had declined in intensity to control values. In contrast, Northern blot analysis of RNA extracted from 18-day old infected MDM cultures (i.e. 25 days post-isolation) exhibited a low level of MnSOD gene expression compared to the uninfected cultures of MDM isolated from the same donor and grown for 25 days (fig. 3).
MONOCYTE/MACROPHAGE Table I. Long-term
RT activity (cpm/ml)
3 5 7 10 12 13 15
1070 990 740 2275 4380 9180 5630
TNFa concentration (m/ml) infected culture uninfected culture supernatants supernatants 30 34 28 36 75 365 102
Quantitative analysis of TNFa mRNA by Northern blot densitometry (arbitrary units) infected uninfected cultures cultures
29 37 40 20 34 36 30
Table shows evaluation of RT activity in the HIV]-infected culture supernatants, TNFa production in infected and uninfected supernatants, quantitative analysis of TNFa mRNA by scanner densitometry of Northern blot (signal intensity was normalized using a constitutively expressed P-actin probe). Results are expressed in arbitrary units.
Uninfected macrophages Fig. 3. MnSOD
were infected with the primary isolate HIVl/DAS which had been preincubated with this bispecific antibody. As table II shows, viral production was greatly inhibited at the three concentrations of bispecific antibody used. As a positive control, we used MDM culture supernatants infected with the viral isolate in the absence of bispecific antibody. Under these conditions, a high level of P24 antigen was detected. Experiments using macrophages pretreated with anti-Leu-3-a mAb and infected under the same conditions as those described above resulted in total inhibition of viral production. In addition to this inhibition, the absenceof P24 antigen production was also observed in the culture supernatants of MDM pretreated with anti-Leu3a mAb and infected with the viral strain HIVl/DAS (table II).
geneexpressionduring the late eventsof
RNA isolated from uninfected and infected macrophagesat 25 days of culture (18 days post-infection) were measuredby densitometricanalysisof Northern blot (signal intensitywasnormalizedusinga constitutivelyexpressed p-actin probe; results are expressed in arbitrary units).
Table II. Measurement of viral replication in MDMinfected culture supernatants using bispecific antibodies composed of anti-gp41 HIV and anti-FcyRI Fab’ fragments. Concentration of bispecific antibodies (ug/ml)
Bispecific antibody activity in infected MDM cultures Bispecific Fab’ anti-FCyRI/gp 41 antibody was usedto test its ability to inhibit viral infection of macrophage cultures. In an initial experiment, MDM
0 19 38 76
P24 antigen (pg/mI) pre-treated no pre-treated anti-Leu3a anti-Leu3a macrophages macrophages 1,175 +_ 275 100 f 40 105* 5 200 * 55
0 0 0 0
Our experimental results suggest that HIVl-infected peripheral blood monocytes/macrophages did not constitutively produce TNFa during the first 10 h of in vitro infection. These results agree with our previous observations (Mabondzo ef al., 1991) and support reports by others that HIV1 infection failed to induce TNFa activity in MDM cultures (Munis et al., 1990). However, our results are in opposition to those of Merill et al. (1989), who described TNFa synthesis lasting from 2 to 12 h after macrophage infection by an HIV1 monocytotropic strain. Here, despite the absence of TNFa production in culture supernatants, we detected TNFa mRNA transcripts 2 and 4 h after infection using Northern blot analysis of RNA extracted from infected macrophage cultures, and TNFa gene expression was significant compared with uninfected cultures. It therefore seems likely that non-specific intracellular signals which might result from viral binding to the macrophage surface could activate low levels of TNFa gene transcription, explaining the absence of TNFa from the supernatants of infected macrophages. During the late events of lentiviral infection, we observed an increase in TNFa synthesis and gene expression at the time when RT activity reached its maximum. The correlation between TNFa production and RT activity observed at a later period of viral infection might be partly due to the intramembranous signals that occur during lentiviral mature particle excretion, which might, in turn, be responsible for cellsurface-associated TNFa production (Krieger et al., 1988). Recent studies demonstrated that TNFa can activate HIV1 expression in cell lines chronically infected with HIV1 (Goldfeld et al., 1991). TNFa synthesis observed at a time of high viral replication might result from a consequence of the presence of secondary intracellular mechanisms during the last stage of viral maturation and might be responsible for the hyperactivation of viral replication. The antimicrobial mechanisms specific to the activated macrophages deriving from the respiratory burst are now well characterized. Reactive oxygen intermediates such as superoxide anion (OF), hydrogen peroxide (Hz03 and hydroxyl radical (OH”) exhibit microbitidal activity against bacterial, parasitic and viral infections. Several authors reported that MDM exert effective microbicidal activity against intracellular parasites by means of intracellular oxygen-dependent mechanisms. However, to regulate the synthesis of these toxic metabolites and to protect MDM against them, oxidative burst activity can be inhibited by scavengers such as SOD or catalase. In the present work, the study of MnSOD gene expression, a SOD exclusively produced in mitonchondria, was of interest because (1) HIV replication in macrophages is
intracellular, and (2) in a recent study, N-acetyl-Lcysteine, an antioxidant molecule, was reported to block HIV replication in vitro (Roederer et al., 1990). Here, we measured a high level of MnSOD gene expression 2 and 4 h after infection. This may have been due to early cellular activation by the HIV infection and might explain the inability of the macrophages to destroy, soon after infection, the intracellular microorganisms present in the macrophages of HIVl-infected patients. In contrast, the decrease in the level of MnSOD gene expression during the phase of high proviral DNA transcription (i.e. 18 days post-infection) reflects impairment of cellular microbicidal activity and an imbalance between oxidant and antioxidant mechanisms. Human monocytes/macrophages express Fey receptors on their membrane surface. Three distinct classes have been described (FcyRI, FcyRII and FcyRIII) and each class of receptor may be considered as a means of monocyte/macrophage infection. Takeda et al. (1990) recently demonstrated that anti-FcyRI mAb are able to block infection of the U937 cell line by antibody-complexed HIVl. The results we observed using bispecific antibody suggest that HIV1 infection of MDM may be inhibited by the interaction of opsonized virus with FcyRI. Furthermore, despite the fact that CD4 molecule are expressed at subthreshold levels on macrophage membranes (below the detection limit of flow cytometry analysis), this infection was completely inhibited by pretreatment of the cells with anti-Leu3a antibody. This finding concords with reports by other authors, who suggested that the infection of MDM requires interaction of the virus with CD4 (Matsuda et al., 1989; Takeda et al., 1990). Taken together, the results referred to above may account for some clinical and biological features observed in man during human HIV infection. Thus, TNFa production may be involved in the clinical symptoms observed in this infection, such as cachexia. Impairment of microbicidal activity might explain the persistence of virions in the MDM. Lastly, the neutralizing activity of bispecific antibody suggests that they might play a positive role in rational immune/antiviral therapy of HIV infection.
Acknowledgements The authors are grateful for the assistance of the Centre de Transfusion Sanguine des Armies (Clamart, France). This work was supported by the Agence Nationale pour la Recherche contre le SIDA (France) and the Fondation Europeenne pour la Recherche sur le SIDA.
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