Effects of colony stimulating factors on the interaction of monocytes and the human immunodeficiency virus

Immunology Letters, 19 (1988) 193-198

Elsevier IML 01108

Effects of colony stimulating factors on the interaction of monocytes and the human immunodeficiency virus M o n t e S. M e l t z e r a n d H o w a r d E. G e n d e l m a n * Department of Cellular Immunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, U.S.A.

(Received21 June 1988; accepted 26 June 1988)

1. Introduction Cells of mononuclear phagocyte lineage are major targets for the human immunodeficiency virus (HIV) in the infected human host. HIV-infected monocytes escape immune surveillance and ultimaterelatively low levels through extended time intervals in the face of an often vigorous, but apparently ineffective host immune response. How these infected monocytes escape immune surveilance and ultimately effect immunodeficiency are questions whose answers are central to understanding the pathogenesis o f the acquired immune deficiency syndrome (AIDS). This report will review (a) evidence for mononuclear phagocytes as in vivo targets for HIV infection, (b) mechanisms that explain how HIVinfected monocyte/macrophages evade a competent host immune surveillance, an evasion that ultimately results in viral persistence, (c) the possible roles of persistently infected monocyte/macrophages in the pathogenesis of AIDS-associated immunosuppression and neuropathy, and (d) the utility o f monocytes as susceptible target cells for the efficient isolation and propagation of virus from patients at the various stages o f HIV disease. Key words: HIV; AIDS; Pathogenesis;Monocyte;Macrophage;

T cells Correspondence to: Monte S. Meltzer,M.D., Dip~ctor,Program

for NonspecificImmunity,Departmentof Cellular Immunology, Walter ReedArmy Institute of Research,Washington, DC 203075100. U.S.A. Tel. (202)-576-2570. * Dr. H. E. Gendelmanis a Carter WallaceFellowof Columbia University College of Physicians and Surgeons.

2. The mononuclear phagocyte as a target cell for the human immunodeficiency virus: lessons from lentivirus infection of sheep and goats The ruminant lentiviruses, e.g. visna and caprine arthritis-encephalitis viruses of sheep and goats, share many biologic, biochemical and molecular properties with their human counterpart, HIV. Perhaps the most important of these shared properties relates to mechanisms and sequelae of viral persistence: infected monocyte/macrophages serve as a viral reservoir, evade host immune surveillance and initiate fulminant disease with inflammatory and/or degenerative changes in the immune and central nervous systems [1]. Lentivirus replication is dependent upon both the state o f maturation and differentiation of the infected cell [2]. In certain tissues o f infected animals, such as bone marrow or blood, the number o f cells (monocytes or their precursors) that express viral RNA is very low. After these infected monocytes migrate from blood and mature into tissue macrophages, viral gene expression increases several thousand-fold. A similar phenomenon occurs in vitro as infected monocytes differentiate into macrophage-like cells with time in culture. Certain genetically predetermined, cellular transcriptional factors (factors that vary with both macrophage maturation and differentiation) may regulate viral gene expression and/or virus cell surface receptors. Such factors are found in the subpopulations of monocyte/macrophages that support viral replication and ultimately provide the molecular basis for the unique tissue tropism that underlies viral pathogenesis and the symptomatology o f lentivirus infections in ruminants and man.

0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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3. The HIV-infected monocyte and the subversion of host immune surveillance

In HIV-infected individuals, virus has been detected by direct isolation, by nucleic acid hybridization, and by immunocytochemical or electron microscopic analysis of: brain macrophages [3, 4], follicular dendritic cells of lymph nodes [5], monocytes in peripheral blood and bone marrow [6, 7], Langerhans cells in skin [8] and alveolar macrophages [9]. Analogous to observations with the ruminant lentiviruses, very few human bone marrow cells or circulating blood leukocytes show evidence of permissive viral replication: the frequency of infected cells in these tissues as detected by in situ hybridization with HIV-specific RNA probes range from 0.001 to 0.0001070 [101. In contrast, brain macrophages express HIV RNA in frequencies as high as 15°70. Indeed, the predominant cell in brain in which HIV gene expression is evident is the macrophage: HIV-specific RNA is detected in macrophages of the subarachnoid, perivascular and white matter parenchyma cell populations. Significantly, virus is not found in neurons or neuroglia (astrocytes or oligodendrocytes) [11]. A similar phenomenon occurs in skin: the frequency of infected Langerhans cells in epidermis may be 10000-times that of blood leukocytes in the same patient. Several potential mechanisms exist during HIV infection that allow the virus of infected cells to escape host immune responses: inadequate levels of neutralizing antibody, antigenic drift (epitope changes in the viral envelope), persistent but restricted infections of long-lived bone marrow stem cells, and others. Studies in our laboratory on the interactions of HIV and monocytes have uncovered yet another mechanism for viral escape from immune surveillance. Electron microscopic analysis of HIVinfected T cells shows literally hundreds of viral particles associated with the plasma membrane: HIV assembles and buds only from the plasma membrane of infected T-cells; there is no intracellular accumulation of mature or even immature virions. HIV interaction with macrophages is quite different from that of T cells (Fig. 1). Ultrastructural analysis of HIV-infected macrophages 10 and 40 days (time intervals where 60 to 90°70 of ceils express both HIVspecific mRNA and proteins) after infection show few or no virions at the plasma membrane [7]. Yet 194

these infected cells contain large numbers of viral particles. Virus is localized almost exclusively to intracellular vacuoles. Infected macrophages display numerous vacuolar structures, unassociated with the plasma membrane, each of which contain scores of mature and immature virions. Indeed, HIV not only accumulates within these intracellular vacuoles but also assembles and buds from vacuolar membranes. Morphologic evidence strongly suggests that these vacuoles are derived from the Golgi of the macrophage [12]. In essence, the macrophage handles HIV virus much like any other secretory glycoprotein: HIV is assembled in the Golgi and transported in Golgi-derived vacuoles toward the plasma membrane. Significantly, the final step of secretion, exocytosis into the extracellular milieu, appears suppressed: the amount of virus released from HIV-infected monocyte/macrophages, quantitated by reverse transcriptase or p24 antigen in culture fluids, is 10-fold less than that released by an equal number of infected T-cells. Thus, the HIVinfected monocyte/macrophage represents a veritable virus factory, but a factory whose entire output remains hidden from the host. Experiments confirm that the intracellular virions of HIV-infected macrophages are infectious: release of these viral particles by freeze-thaw cycles increases the infectious titer of the culture fluids by at least 10-fold [7]. Most importantly, these in vitro observations have been confirmed in the AIDS patient. Macrophages in the brain of a seropositive individual also show intracellular localization of virus particles within vacuoles; little or no virus was detected at the plasma membrane [12]. Such virus, sequestered from host immunity within cytoplasmic vacuoles, represents a true reservoir for continued infection. Release of infectious virus from this macrophage reservoir and dissemination of HIV into other macrophages or T ceils could be initiated by any agent that perturbs macrophage function: factors released during inflammation, normal tissue remodeling or host response to intercurrent infection. 4. The role of the HIV-infeeted monocyte in the pathogenesis of AIDS

The preceding observations clearly document a role for monocyte/macrophages as both target cell and reservoir for infectious virus during HIV dis-

o

Fig. 1. Transmission electron microscopy of (left panel) an HIV-infected MCSF-treated monocyte (viral particles sequestered within intracytoplasmic vacuoles; few or no virions at the plasma membrane) and (right panel) PHA/IL-2 treated lymphoblasts (numerous viral particles budding at the plasma membrane; no intracellular virions) x 9,200 [7, 12]. (The authors thank Dr. Jan M. Orenstein, Department of Pathology, George Washington University Medical Center, Washington, DC for electron microscopy.)

ease. HIV-infected monocytes/macrophages are found in brain, lung, lymph node, skin, bone marrow and blood of seropositive patients. In certain tissues, notably brain and skin, the frequency of infected cells approaches 1 in 10. It is probable that these infected cells directly participate in the pathogenesis of HIV-induced immunosuppression and central nervous system dysfunction. However, the means and mediators of this participation are not well understood and much of the current evidence for macrophage dysfunction is conflicting [13, 14]. A major role of monocyte/macrophages during the steadystate and inflammation is regulation of tissue function. This regulatory role is mediated by the literally hundreds of secretory molecules released by the macrophage under a variety of pathophysiologic conditions [15]. Changes in the secretion or release of certain mediators occurs during HIV infection and underlies the symptomatology of AIDS. The paucity of virus-infected lymphocytes in AIDS and absence of cytolytic infections of neurons or neu-

roglia suggest such an indirect mechanism for immune and nervous system dysfunction in HIV infection. Indeed, recent studies in our laboratory suggest that disordered secretion of one or more monokines may initiate much of the central and peripheral neuropathy of AIDS [16]. AIDS-associated encephalopathy is characterized by vacuolar degeneration of white matter in the absence of significant inflammation coincident with the appearance of numerous astrocytes, foamy perivascular macrophages and multinucleated, macrophage-derived giant cells. Monocyte/macrophages treated with macrophage colony stimulating factor (MCSF; Cetus Corp., Emeryville, CA) and granulocyte/macrophage colony stimulating factor (GMCSF; Immunex Corp., Seattle, WA) secrete a neurotrophic factor that promotes growth and differentiation of neurons in culture. The effects of this monocyte-derived, neurotrophic factor cannot be simulated by the conventional, lymphokine-rich, culture fluids of mitogen-stimulated T cells or by recombinant hu195

man interleukins 1 through 6, interferons ct or -/, granulocyte colony stimulating factor (GCSF), MCSF or GMCSF, tumor necrosis factor (TNF)a, transforming growth factor/3, or fibroblast or nerve growth factors. Release of this monocyte-derived neurotrophic activity is abrogated by HIV infection. Concurrent with the loss of neurotrophic activity, HIV-infected monocytes increase secretion of another activity, a factor that is directly toxic to neurons. This neurotoxic activity is not a direct effect of HIV itself. Culture fluids from HIV-infected T-cell cultures, purified high-titer virus (LAV) or the envelope glycoprotein, gpl20, had no toxic effect. The toxic activity in culture fluids of HIV-infected monocytes was unaffected by -/-irradiation sufficient to neutralize HIV or by immunoaffinity adsorption with solid-phase seropositive AIDS patient sera. The HIV-induced, monocyte-derived, neurotoxic activity is relatively selective in its target cell: neurons are exquisitely sensitive to the toxic effect, while fibroblasts remain unaffected and brainderived astrocytes paradoxically proliferate. In sum, the in vitro effects of this monocyte-derived neurotoxic factor (and the virus-induced loss of monocyte neurotrophic activity) remarkably mimic the pathology found in AIDS-associated encephalopathy: neuronal degeneration and death with coincident proliferation of astrocytes around foci of HIVinfected macrophages. Similar toxic factors may also be involved in the depletion of CD4 ÷ T cells, the hallmark of AIDS. Productive HIV infection is rare in blood leukocytes suggesting that the mechanism(s) of T4 lymphocyte depletion involves events other than direct, viralinduced cytopathogenicity. Toxic factors secreted by HIV-infected monocytes may work in tandem with other mechanisms (toxic effects of HIV envelope glycoproteins or destruction of infected T cells by autoimmune phenomenon) to effect depletion of the T4 cell [17]. 5. Monocytes as target cells for efficient isolation and propagation of HIV from infected individuals

The ability to isolate virus from patient blood onto mitogen-stimulated lymphoblast cultures (the conventional T cell isolation of HIV) increases with stage of HIV disease: virus isolation is successful in about 20 to 30°70 of seropositive, asymptomatic pa196

tients and increases in frequency to about 80°-/oof patients with frank AIDS. We recently reported a technique to isolate HIV into cultured human monocytes [7]. Relatively pure populations of monocytes are obtained by countercurrent centrifugal elutriation of mononuclear leukocyte-rich fractions of blood cells from normal donors undergoing leukophoresis. This procedure results in cell suspensions _> 96°70 monocytes by criteria of morphology on Wrightstained cytosmears, by granular peroxidase or by nonspecific esterase. Such purified monocytes can be cultured for intervals _>3 months in medium supplemented with human serum and recombinant human MCSE These cells provide susceptible target cells for HIV infection: cocultivation of peripheral blood mononuclear leukocytes (PBML) from seropositive individuals and MCSF-stimulated monocytes from normal donors resulted in isolation of progeny HIV virions in virtually all patients tested. Monocytes pretreated with MCSF for one week were used for cocultivation experiments with freshly isolated PBML from seropositive individuals. By FACS flow cytometry at one week > 98 °7oof purified MCSF-treated monocytes were positive for HLe-1 (CD 45) and Leu-M3 (CD 14), but binding of antiB4 (CD 19), T4 (CD 4), T6 (CD1), T8 (CD 8) or Tll (CD 2) were each below levels of detection. Aliquots of PBML from each of 27 patients were cocultivated with MCSF-treated adherent monocytes and suspensions of PHA/IL-2 stimulated PBML (lymphoblasts) from normal donors. Culture fluids were sampled daily and assayed for HIV-specific antigens and reverse transcriptase (Table 1). Isolation of HIV onto MCSF-treated monocytes was successful in 25/27 patients (93070). In contrast, virus isolation from replicate aliquots onto mitogen-stimulated lymphoblasts was successful in only 11/27 attempts (4107o). It is important to note that unlike the virus isolation into T cells, recovery of HIV into MCSFtreated monocytes was equally successful in both early and late stages of disease. Indeed, several reports show that recovery of HIV into monocyte/macrophages may be the solely successful virus isolation system in early disease: in a seropositive patient with laboratory-acquired HIV infection or in several seronegative patients with acute HIV infection, virus was isolated from blood only into cultured monocytes, not T cells [18, 19].

TABLE 1 Virus isolation from blood leukocytes of patients at risk for or infected with HIV [7]. WR stagea

0 1/2 3/4 5/6 Total

Serotype

neg pos pos pos

Number of patients

2 9 3 13 27

Patient leukocytes added to MCSF-treated monocytes

PHA/IL-2 treated lymphoblasts

2/ 2 7/ 9 3/ 3 13/13 25/27 (93°7o)

O/ 2 2/ 9 2/ 3 7/13 11/27 (41%)

a Walter Reed staging classification for HIV infection [seereference 21]: stage 0 (seronegativeindividuals with known risk factor); stage 1/2 (seropositiveminimallysymptomatic patients); stage 3/4 (T cell depletion without opportunistic infection); stage 5/6 (frank AIDS).

Progeny virions released in culture fluids of infected monocyte and lymphoblast cultures were used to serially infect other MCSF-treated monocyte and P H A / I L - 2 stimulated lymphoblast cultures: m o n o cyte tropic-HIV isolates were serially passaged onto other MCSF-treated monocytes; T cell tropic-HIV isolates were serially passaged onto other P H A / I L - 2 stimulated T cell cultures. Monocyte and T celltropic H I V isolates were also added to heterologous cells (monocyte-tropic H I V onto lymphoblasts and T cell-tropic H I V onto monocytes). In these cultures, sustained, productive virus infection was not demonstrated. Moreover, several different strains of HIV, such as LAV, that were each maintained for long intervals in normal lymphoblasts or continuous T cell lines all failed to infect MCSF-treated m o n o cyte/macrophage cultures even at viral inocula 20fold higher than that needed to infect lymphoblasts. Thus, we document a restricted biologic tropism a m o n g H I V isolates [7]. In a single patient we are able to recover 2 H I V isolates; a monocyte-trophic variant that infects and replicates within MCSFmonocytes and a T cell-tropic variant that infects and replicates within P H A / I L - 2 stimulated T cells. For each H I V variant, infection of the heterologous cell type is abortive or inefficient. The proliferative capacity o f MCSF-treated monocytes is low. At any one time, the number of actively dividing cells in these cultures ranged from 1 to 3°7o o f the total. This frequency is similar to that reported for tissue macrophage populations in the steady-state. The number of actively dividing mono-

cytes was markedly increased by the addition of another macrophage growth factor, recombinant hum a n G M C S F [20]. Incorporation of [3H]thymidine into macrophage populations treated with MCSF and G M C S F was increased 20-fold over that of cells treated with either growth factor alone. There was little or no cell proliferation of monocyte/macrophages with _< 1000 U / m l MCSF or 4000 U / m l G M C S F alone. Optimal levels o f cell proliferation were detected at 1000 U / m l MCSF and 50 U / m l GMCSF. H I V infection and replication within monocyte/macrophages treated with MCSF and G M C S F was very different from that of cells treated with either growth factor alone. After an identical virus inoculum, levels of fluid-phase reverse transcriptase activity and p24 antigen in cultures of H I V infected macrophages treated with both growth factors (MCSF + GMCSF) were more than 3-fold greater than those of cells treated with MCSF alone. Peak levels of reverse transcriptase were detected earlier (9 days) in monocyte cultures treated with M C S F + G M C S F than in cultures treated with MCSF alone (13 days). The most obvious difference between these infected macrophage cultures was in HIV-induced cytopathogenicity: HIV-infected, MCSF-treated monocytes display marked cytopathogenic changes with infection; the addition o f 50 U / m l G M C S F completely abrogated these virus-induced changes. Thus, release of virus by HIV-infected, M S C F + G M C S F - t r e a t e d macrophages occurs earlier and at higher levels than that by infected cells treated with MCSF alone. Cells 197

t r e a t e d with b o t h M C S F a n d G M C S F proliferate in culture a n d show little o r n o evidence o f H I V i n d u c e d c y t o p a t h o g e n i c c h a n g e t h r o u g h 40 days o f culture. O n c e again, o b s e r v a t i o n s o n H I V m a c r o p h a g e interactions are consistent with similar o b s e r v a t i o n s in lentivirus systems: t h e p e r m i s s i v i t y o f m o n o c y t e / m a c r o p h a g e targets to virus replicat i o n varies with c h a n g e s in cell m a t u r a t i o n a n d / o r differentiation. It is i m p o r t a n t to f u r t h e r n o t e t h a t G M C S F is a c y t o k i n e released by a c t i v a t e d T cells. Levels o f virus r e p l i c a t i o n in infected m a c r o p h a g e s e x p o s e d to G M C S F at sites o f a n i m m u n e r e a c t i o n m i g h t d r a m a t i c a l l y increase, a n d increase in t u r n the p r o b a b i l i t y o f virus infection o f the responsive C D 4 ÷ helper T cell. T h e p r e c e d i n g o b s e r v a t i o n s suggest t h a t the m a c r o p h a g e is the earliest cell infected with H I V a n d f u n c t i o n s as a viral reservoir t h r o u g h o u t disease. T h a t m a n y b o d i l y fluids (semen, c o l o s t r u m , rectal a n d v a g i n a l m u c o u s ) in the s t e a d y - s t a t e h a r b o r m a c r o p h a g e s as n o r m a l cellular c o n s t i t u e n t s (not T cells) suggests t h a t these viral reservoirs m a y directly c o n t r i b u t e to s p r e a d o f disease. But H I V m a y be s p r e a d b y either T cell o r m a c r o p h a g e . Viral replicat i o n in C D 4 + T cells u l t i m a t e l y l e a d s to a d e p l e t i o n o f these cells, a d e p l e t i o n m e d i a t e d by direct viral c y t o p a t h i c effects o r b y h o s t i m m u n e responses a g a i n s t p r o d u c t i v e l y infected cells. Virus persists t h r o u g h s e q u e s t r a t i o n in t h e m a c r o p h a g e , h i d d e n f r o m h o s t i m m u n e surveillance w i t h i n i n t r a c e l l u l a r vacuoles. These c h r o n i c a l l y infected m a c r o p h a g e s initiate i m m u n e a n d n e r v o u s system d y s f u n c t i o n t h r o u g h d i s o r d e r e d secretory f u n c t i o n . A t s o m e p o i n t a n event occurs (an event i n i t i a t e d by intercurrent infection, tissue r e m o d e l i n g , o r n e o p l a s t i c change) t h a t s t i m u l a t e s p r o g e n y virus p r o d u c t i o n by the m a c r o p h a g e o r a m u t a t i o n t h a t changes the m a c r o p h a g e t r o p i c H I V to a T cell t r o p i c virus. Release o f T cell t r o p i c virus repeats the cycle: p r o d u c t i v e l y infected T cells are e l i m i n a t e d by h o s t i m m u n i t y ; the m a c r o p h a g e persists as a reservoir. This e p i s o d i c cycle recurs with progressive a n d relentless T cell d e p l e t i o n to levels t h a t allow o p p o r t u n i s t i c infection a n d AIDS.

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