Macrophages, Multinucleated Giant Cells, and Apoptosis in HIV+Patients and Normal Blood Donors

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 82, No. 2, February, pp. 102–116, 1997 Article No. II964269

Macrophages, Multinucleated Giant Cells, and Apoptosis in HIV/ Patients and Normal Blood Donors BEATRIZ RUIBAL-ARES, NORMA E. RIERA,

AND

MARI´A M. E.

DE

BRACCO

Instituto de Investigaciones Hematologicas, Academia Nacional de Medicina, Buenos Aires, Argentina

INTRODUCTION

Clearance of apoptotic debris is carried out by cells of the monocyte/macrophage lineage and, as other macrophage functions, it can be altered in AIDS, leading to the accumulation of apoptotic cells observed in this disease. In this study we evaluated the ability of macrophages from human immunodeficiency virus (HIV)-infected patients to differentiate and to clear apoptotic debris in prolonged in vitro cultures. Peripheral blood mononuclear cells (PBMC) from infected hemophilia patients were cultured in the absence of exogenously added stimulators and the organization and morphological characteristics of the cultures were analyzed and correlated with clinical staging of the patients. Cell aggregates of different sizes involving macrophages and lymphocytes were formed in cultures from asymptomatic HIV/ patients (CDC groups II–III) and controls and in 4/7 group IV C2 HIV/ patients. In order to obtain viable and organized cultures, cells had to be handled carefully, allowing contact and undisturbed sedimentation in round-bottom tubes. Multinucleated giant cells (MGC) were formed through macrophage fusion after 5 days of culture in HIV0 controls, group II and III patients, and some of the group IV C2 patients, while scarce formation of MGC was observed in AIDS patients or patients with advanced HIV disease. This paucity was correlated with impaired dead cell removal and accumulation of apoptotic debris. Viability of macrophages and MGC was reduced after 15 days. MGC and the macrophages (either free or in cell aggregates) were able to remove dead cells, clearing the cultures of cell debris. Furthermore, in group II and III HIV/ hemophilic patients, increased macrophage–MGC phagocytic activity, suggesting in vivo activation, was frequently observed. In HIV/ patients with AIDS or advanced HIV disease (CDC groups IV A, IV C1, and IV D) dead cell removal was impaired and apoptotic debris accumulated. Long-term cultures of unstimulated PBMC are an interesting model for studying the role of macrophages and/or MGC in the removal of dead cells as well as examining the cellular milieu in which HIV replicates in an individual host. q 1997 Academic Press

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0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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Loss of lymphocyte viability is a frequent observation in peripheral blood mononuclear cells (PBMC) cultures from human immunodeficiency virus (HIV)-infected patients (1). Programmed cell death involving the apoptotic process is thought to play a role in this process (2). Apoptosis is usually distinguished from necrotic cell death by a series of unique cellular changes (3–5). Thus, apoptotic and necrotic dead cells can be distinguished microscopically by their morphology. Diminished cell volume, nuclear retraction, chromatin condensation, and nuclear fragmentation characterize apoptotic cells, while in necrotic cells, damage to the plasma membrane induces osmotic swelling and cell disruption (3–5). In apoptotic cells, activation of nuclear endonucleases induces a ‘‘ladder’’ electrophoretic pattern of oligonucleotide bands, while DNA from necrotic cells yields a continuous smudge of DNA fragments (2). Apoptosis is considered to be a physiologic mechanism of cell removal and, as such, it does not induce an inflammatory response (6). In contrast, necrotic cell death leads to disintegration of the cell membrane with loss of lysosomal enzymes and evokes an inflammatory response (7). Adequate removal of apoptotic debris by macrophages is central to avoiding inflammation and its consequences. Macrophages recognize, phagocytize, and digest both viable and nonviable apoptotic cells, clearing the system of potentially deleterious material (8). Other macrophage-derived phagocytic cells, such as the multinucleated giant cells (MGC) present in granuloma sites and lymph nodes (9– 12), could also contribute to clearance of internalized particles, apoptotic cells, and debris. Whenever an increase in circulating apoptotic cells is observed, it is convenient to assess the ability of phagocytic cells to clear the remains of the apoptotic process. For instance, in HIV infection, increased numbers of apoptotic cells are found in symptomatic patients (13–16) and apoptosis is thought to play a role both in the CD4 and CD8 death and in the anergy observed in AIDS (1, 2). We have recently shown that the apoptotic index (17)

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TABLE 1 Cells/mm3 He

AZT/DDI DDC

CDC

Patient

HIV

T CD4

Monocytes

1 2 3

/ / /

A A A

— — —

II II II

830 1211 665

170 154 511

4 5 6 7 8 9 10 11 12 13 14 15

/ / / / / / / / / / / /

A A A B A A A B A B A A

DDI AZT/DDC AZT/DDC DDI — — DDI DDI AZT DDI AZT AZT/DDC

III III III III III III III III III III III III

430 567 350 540 373 438 550 600 420 470 390 320

122 160 342 60 85 258 366 51 290 472 184 88

16 17 18 19 20 21 22

/ / / / / / /

A A A A B A A

AZT/DDC AZT/DDI AZT/DDC AZT/DDC AZT AZT AZT/DDC

IV IV IV IV IV IV IV

740 124 260 306 377 200 190

402 240 222 234 300 86 108

23 24 25 26 27 28 29 30 31

/ / / / / / / / /

A A A A A B A A A

AZT/DDC AZT/DDC AZT/DDC DDI AZT/DDC AZT/DDC DDI AZT/DDC AZT/DDC

IV A IV A IV A IV A IV A IV A IV A IV A iV A

203 40 252 336 262 394 210 316 380

78 152 144 240 160 172 372 306 72

32 33 34 35 36

/ / / / /

B A A A A

AZT/DDC AZT/DC DDI AZT —

IV IV IV IV IV

75 5 37 60 5

210 282 354 98 92

37

/

A

—

IV D

74

144

38 39 40 41 42 43

0 0 0 0 0 0

A A A A A A

— — — — — —

462 470 1060 1220 620 840

— 148 68 470 205 236

C2 C2 C2 C2 C2 C2 C2

C1 C1 C1 C1 C1

Clinical aspects, OI

HZ HZ VL HZ OC, VL VL ATC

VL, ATC VL

BT, EC PCP, TBC GC, PCP TBC, H NHL, PCP, OC, MH Chagas HTLV-I

Note. He, type of hemophilia (A, Factor VIII deficiency; B, Factor IX deficiency); HZ, herpes zoster; VL, villous leukoplasia; OC, oral candidiasis; ATC, atrophic tongue candidiasis; BT, brain toxoplasmosis; EC, esophageal candidiasis; H, hystoplasmosis; PCP, pneumocystis carinii pneumonia; GC, generalized candidiasis; TBC, tuberculosis; NHL, non-Hodgkin lymphoma; MH, mucocutaneous herpes; Chagas, positive Chagas disease serology; HTLV-I, positive HTLV-I serology.

of PBMC from HIV/ hemophilic patients increased with the severity of HIV disease and with disease progression (16). Microscopic observation, corroborated by DNA electrophoresis and colorimetric determination of

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soluble DNA fragments, demonstrated that apoptosis was the main form of lymphocyte death in AIDS PBMC cultures (16). It is not known if the increased apoptotic index reflected augmented apoptosis or failure to re-

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move apoptotic cells, or if it was the result of both processes. Since removal of apoptotic cells and apoptotic bodies is carried out mainly by macrophages, impaired ability of macrophages to remove apoptotic cells could underly their accumulation in AIDS (18). Although most of the discussion on the role of apoptosis in HIV infection has been centered on lymphocyte depletion, insufficient clearance of dead cells and apoptotic debris by overwhelmed macrophages could contribute to impairment of the regulation of the immune system in AIDS patients (18, 19). In order to study the factors that affect the balance of lymphocyte function in HIV-infected patients, it is convenient to reproduce in an in vitro system the milieu in which the interactions between HIV, T lymphocytes, and macrophage-derived cells take place. Undisturbed culture of PBMC for up to 25 days, without exogenous cytokine additions or allogeneic cell coculture, proved to be a simple system with which to study the ability of macrophages and macrophage-derived MGC from HIV-infected hemophilic patients to remove apoptotic lymphocytes and maintain culture viability. Our results demonstrate that in vitro generation of MGC and survival of macrophages are greatly impaired in advanced HIV-infected patients and AIDS patients. In PBMC cultures from these patients, dead cells were increased in comparison to those from HIV 0 controls. Furthermore, absence of MGC after 6 – 10 days of culture correlated with the loss of culture viability and accumulation of cell debris, suggesting that in addition to macrophages, in this experimental model MGC participate in the remotion of apoptotic cells and debris. MATERIALS AND METHODS

Patients Thirty-seven hemophiliac patients (age 23.3 { 1.3 years) who were periodically evaluated for clinical and

laboratory signs of HIV disease progression and/or antiretroviral therapy and who received assistance at the Fundacio´n Argentina de Hemofilia were included in this study. Patients who had undergone major surgery or received blood transfusions in the 6 months prior to this study were excluded. Since 1985 all patients had received heat-inactivated commercial concentrates of intermediate purity (Factor VIII for hemophilia A and Factor IX for hemophilia B patients) (20). Serologic evidence of HIV infection was obtained by two independent ELISA tests (Abbott) and confirmed by Western blot or indirect immunofluorescence (21). A summary of the clinical data is shown in Table 1. Patients were classified according to the CDC (22): group II, asymptomatic HIV infection (n Å 3); group III, persistent generalized lymphoadenopathy (n Å 12); group IV A, HIV-related constitutional disease (n Å 9); IV C1, specific secondary infections listed in the CDC surveillance definition of AIDS (n Å 5); group IV C2, other specified secondary infectious diseases (n Å 7); group IV D, secondary cancer (n Å 1). Around 80% of the patients had positive hepatitis B (HBV) or C (HCV) serology. This high frequency of HBV and HCV positivity was similar to that in HIV/ and in HIV0 hemophiliacs. One of the HIV0 patients had positive serology for Chagas disease (anti-Trypanosoma cruzi) and one had positive HTLV-I serology. Controls were HIV-, HBV-, HCV-, HTLV-I-, and Chagas disease-negative volunteer blood donors. Purification of PBMC, CD4 Determination, and Macrophage Identification PBMC were obtained by Ficoll–Hypaque centrifugation of heparinized blood (23). CD4 lymphocytes were assayed by flow cytometry of whole blood after lysis with FACS lysing reagent using FACScan (Becton– Dickinson) equipment and the SIMULSET program. Anti-CD3 (Leu4), anti-CD4 (Leu3), and anti-CD8 (Leu2) monoclonal antibodies (Becton–Dickinson, Bio-

FIG. 1. Macrophages, multinucleated giant cells (MGC), and apoptosis in PBMC cultures of HIV/ patients. Preparations from patients and controls were stained with acridine orange and ethidium bromide as described under Materials and Methods. Examples from individual patients were chosen to illustrate the different aspects and stages of PBMC culture organization according to the patterns defined in Table 2. (A) Macrophages engaged in mitosis (central arrows) in an intermediate size cell clump. Right arrow points at a macrophage containing ingested and partially digested apoptotic material (6-day PBMC culture from a normal donor). Original magnification, 4001. (B) MGC containing phagocytized degraded material (arrow); macrophages containing phagocytized debris are also shown (9-day PBMC culture from patient 11, group III). Original magnification, 2501. (C) MGC showing viable green-stained zones and a nonviable sector stained in orange (arrow at the bottom, right). Arrow at the top, right, signals a prolongation of the MGC in close contact with a binucleated macrophagederived cell. MGC contains partially degraded apoptotic material (7-day PBMC culture of patient 9, group III). Original magnification, 2501. (D) Macrophage clump showing macrophages in the process of fusion (top right) and dead macrophages adhering to amorphous material. A small cell aggregate including a macrophage (or dendritic cell?) rosetting with five viable lymphocytes is shown on the left side (17-day PBMC culture of patient 6, group III). Original magnification, 2501. (E) Final organization of intermediate cell aggregates including viable (right arrow) and nonviable MGC (left arrow). A core of amorphous material is shown in the center of the clump (central arrow). A small cell clump is shown in the top of the preparation. Clean background with few free orange-stained dead cells (12-day PBMC culture of patient 13, group III). Original magnification, 1001. (F) Unorganized PBMC culture showing abundant orange–red-stained dead lymphocytes and green-stained viable lymphocytes with apoptotic nuclei. Few macrophage aggregates. The arrow points to one viable vacuolated macrophage (5-day PBMC culture of patient 32, group IV C1). Original magnification, 2501.

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systems, Argentina) were used (24). Monocytes were determined by flow cytometry using the Cell Quest program and CD45/CD14 monoclonal antibodies (Becton– Dickinson). Cells of the macrophage line were identified by indirect immunofluorescence using monoclonal anti-human macrophage antibodies (CD68, clone K P1, DAKO) and fluorescent-labeled anti-mouse IgG (Fab2 anti-mouse, Cappel). Assay of HLA class II antigens (DR) and CD11c on MGC was carried out by direct immunofluorescence using monoclonal antibodies (fluorescent-labeled anti-HLA-DR and CD11c, Becton– Dickinson). In all cases, cells recovered from cultures were handled gently to avoid rupture of the aggregates. More than 200 cells were counted. Viability was checked by trypan blue exclusion and morphology by May–Grunewald–Giemsa staining: pellets were suspended in 25 ml RPMI 1640 tissue culture medium with 10% fetal calf serum (GIBCO, Grand Island) and 8.5 mg% gentamycin (RPMI–FCS). Out of the 25 ml of suspended pellets, 12 ml was extended carefully on a glass slide and evaporated at room temperature avoiding complete dryness and alteration of cell morphology. Preparations were fixed with methanol–acetone mixture (1/1, v/v) for 5 to 10 min, washed 2–3 times with pH 7.4 phosphate-buffered saline (PBS), and covered with May–Grunewald solution for 1 min; 0.5 ml of distilled water was added and left for an additional 2–3 min. Then the stain was discarded and the slide was covered with diluted Giemsa reagent (1 drop/2 ml) and stained for 15 min.

Assay of apoptosis was carried out as described previously (16) by measuring the differential uptake of two nucleic acid-staining fluorescent stains: acridine orange, which penetrates viable cells staining the nuclei green and cytoplasmic RNA red; and ethidium bromide, which stains the nuclei of nonviable cells orange– red. Cells were considered apoptotic when they presented the changes that characterize the different stages of the apoptotic process, namely reduction of cell volume with blebbing and zeiosis, cellular retraction with nuclear chromatin collapsed in crescents, fully collapsed nucleus, or nucleus broken into condensed spheres and apoptotic bodies. In brief, 25 ml of the resuspended culture pellet was stained with 1 ml of 100 mg/ml acridine orange (Sigma) / 100 mg/ml ethidium bromide (Sigma) solution. For microscopic observations, 10 ml of this cell suspension was placed on a slide and carefully covered. Most unaggregated cells were displaced to the periphery, while the bigger cell clumps remained in the center of the preparation. Thus, in order to examine the organization of cell accumuli, the central fields were studied and observed microscopically with a Zeiss epifluorescence microscope using a halogen 6V, 15-W UV lamp. The apoptotic index (AI) of lymphocytes was calculated after 48 hr of culture without mitogen stimulation, as described previously (16, 17).

PBMC Cultures

Culture Conditions, Cell Organization, and Generation of MGC

PBMC cultures were carried out in round-bottom 5ml polystyrene tubes (Falcon) containing 2 1 106 PBMC that were suspended in 2 ml RPMI–FCS. The tubes were left in a vertical position throughout the culture period. Beginning on Days 5–6 of culture, partial changes in RPMI–FCS (1 ml every 3–4 days) were made without centrifugation by gentle aspiration above the cell pellet. During the first hours of culture, PBMC settled to the bottom of the tube, forming a compact pellet. For microscopic observation this pellet could be easily aspirated with a plastic tip, avoiding disturbance of the supernatant. Because cells rolled to the bottom and packed in the pellet, very few cells remained adhered to the tube’s walls after pellet removal. For each patient 3–12 different tubes were set up and left undisturbed in the CO2 incubator in order to observe cell aggregates and the formation of MGC. Cell aggregates are clumps of different sizes containing monocytes/ macrophages and some adhered lymphocytes. MGC are cells containing more than three nuclei englobed by the same plasma membrane.

Unstimulated PBMC cultures from normal donors (N-PBMC), HIV0, and HIV/ hemophiliacs (HIV0 PBMC and HIV/ PBMC) were observed for up to 25 days. The acridine orange–ethidium bromide tinction was routinely used. May–Gru¨mwald–Giemsa-stained preparations were also examined. After 18 hr, small clumps of monocytes could be observed in N-PBMC, HIV0 PBMC, and 14 of the 37 HIV/ PBMC studied. These cell aggregates increased their size and both viable and nonviable lymphocytes were incorporated (Fig. 1A). After 3–4 days of culture, phagocytosis of dead cells, apoptotic bodies, and cell debris by free macrophages or macrophages from the cellular aggregates became evident (Fig. 1A) and increased with the time of culture, reaching a plateau that varied in the different PBMC cultures (7–15 days) as the number of viable lymphocytes dropped slowly. Viable lymphocytes without the nuclear signs of an ongoing apoptotic process also became attached to, or came into close contact with, these macrophage clumps (Figs. 1A and 1D). In normal controls and in patients belonging to

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Morphological Assay for Apoptosis

RESULTS

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MACROPHAGES, MULTINUCLEATED GIANT CELLS, AND APOPTOSIS IN AIDS

groups II and III, as well as in a few of those in groups IV C2 and IV A, MGC started to form after 5 to 6 days of culture, mainly in the periphery of the cell aggregates and contained phagocytized material. Nuclei appeared to be evenly distributed in these polykaryons. MGC came in close contact both with free or aggregated macrophages and with viable or nonviable lymphocytes (Fig. 1C). Fifty percent of the MGC were found within the macrophage aggregates but free MGC of different sizes and viability could also be observed. Viability of these MGC decreased with time of culture. In a given preparation large and small macrophage aggregates and viable MGC at different stages of formation coexisted with nonviable MGC or free macrophages (Figs. 1B, 1C and 1E). The aspect of cell aggregates was typical: lymphocytes in variable number and phagocytic cells (many of them engaged in the phagocytosis of apoptotic material) were located at the periphery while an amorphous substance formed the central core of the clump (Figs. 1D and 1E). Viable and nonviable MGC were intercalated with macrophages in the external part of the clumps. Mitotic figures (Fig. 1A) and macrophages in the process of fusion (Fig. 1D) were frequently seen in cell aggregates around Days 5–7. Fusion of macrophages at different stages of maturation gave rise to MGC and was preceded by close contact between the plasma membranes leading to the formation of cytoplasmic bridges. MGC in the periphery of the clumps were actively engaged in apoptotic cell removal (Fig. 1B), but MGC outside the cell aggregates could also trap other cells containing apoptotic material (Fig. 1C). Between 6 and 15 days of culture cell clumps reached their maximum size (large aggregates: 450–600 mm) and could occupy 70–80% of the microscopic field at 1001. The most frequently observed cell aggregates were about half that size (intermediate aggregates: 200–400 mm) and small clumps (100–150 mm) were also observed. The final morphological stage of these organized cell clumps also began to appear at 4–6 days and involved the formation of an amorphous material released from macrophages engaged in the phagocytic process. This material attached to most cells within the clump except the peripheric lymphocytes (Figs. 1D and 1E). Finally, after 15–18 days of culture, both the number and the size of the cell aggregates decreased. In contrast, PBMC cultures from patients with advanced HIV infection or AIDS failed to organize, macrophages did not remove or process apoptotic cells, and the general aspect of the PBMC was quite different: macrophages were virtually absent or damaged at Days 5–6, nonviable apoptotic lymphocytes accumulated and no MGC were formed (Fig. 1F). Figure 2 shows the morphology typical of emerging MGC in May–Gru¨mwald–Giemsa-stained smears of 7to 20-day PBMC cultures. A lymphocyte with con-

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densed nucleus is already inside the MGC and another lymphocyte adheres to the MGC membrane (Fig. 2A). A macrophage linked to the same MGC by a narrow bridge is shown in Fig. 2B. In Fig. 2C, a MGC with six nuclei and two lymphocytes trapped by surface microvilli is shown. The phenotype of the phagocytic cells that organized into aggregates or formed MGC was established by staining with CD68, CD11c, and anti-HLA class II antibodies (Fig. 3). Figure 3A shows intermediate-sized clumps stained with anti-HLA-DR and Fig. 3B shows the same preparation stained with anti-CD11c. Likewise, cell aggregates were stained with CD68 (not shown), suggesting that activated macrophages integrated the cell aggregates. The macrophage lineage of MGC was confirmed by their reaction with anti-CD68, anti-HLA-DR, and anti-CD11c. Figure 3D shows an MGC with its membrane stained with anti-CD68, and in Fig. 3C the same MGC is shown upon phase contrast. Anti-CD68 stain was more brilliant in membrane zones devoid of blebs. Taken together these results confirm that activated macrophages organize into cell clumps upon culture and give rise to MGC mainly through fusion. In addition to activated macrophages, other cells with dendritic cell morphology could be observed in PBMC cultures (data not shown). The characteristic organization of the cell cultures described above could be observed only when whole PBMC of normal donors or HIV/ patients in the less severe stages of the disease were used. The shape of the culture tubes was important, as the formation of cell aggregates and MGC was observed only when round-bottom tubes were used. Flat-bottom multiwell dishes resulted in scarce formation of small cell aggregates and few MGC containing less than four nuclei. Separation of nonadherent from adherent cells by adhesion to plastic dishes prevented the formation of large cell aggregates or MGC. Furthermore, clearance of dead cells, cell debris, and apoptotic bodies was seen only in unfractionated cultures of normal PBMC or PBMC from group II and group III HIV/ patients. PBMC Culture Characteristics and HIV Disease As described previously (16), apoptosis was high in 48-hr unstimulated PBMC cultures from symptomatic HIV/ patients (AI%: group II, 18 { 6; group III, 21 { 6; group IV C2, 35 { 4; group IV A, 43 { 4; group IV C1, 63 { 5; group D, 70; HIV0 hemophiliacs, 15 { 7; normal controls, 11 { 3). Therefore, we continued to observe such cultures after 7–25 days, trying to correlate the morphology of macrophages and macrophagederived phagocytic cells with culture viability and clearance of dead cells and/or apoptotic debris. In order

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TABLE 2 Classification of PBMC Cultures According to Their Organization and Morphology Culture pattern

Remotion of AC Mono/M aggregates Large Intermediate Small MGC Viable lymphocytes Viable apoptotic or nonviable lymphocytes Free apoptotic bodies

a

b

c

d

e

f

g

h

5/

5/

4/

3/

2/

/

—

—

5/ — — 5/ 4/ — —

4/ — — 4/ 5/ { —

3/ — — 3/ 5/ / —

3/ — — 3/ 3/ 2/ —

— 3/ — 2/ 2/ 3/ {

— 3{ 2/ — / 2/ 2/

— — { — { 2/ 2/

— — — — — 3/ 3/

Note. On the basis of the morphology of PBMC cultures from HIV-infected patients (n Å 37) and normal controls (n Å 50), a score of 0 to 5/ was established for different items defining the viability and organization of the cultures, the ability of macrophages to generate multinucleated giant cells (MGC), and their ability to remove apoptotic cells (patterns a–h). The organization and morphology of most normal PBMC cultures (40/50) at the plateau period (7–15 days) were considered standard (pattern c). Preparations were stained with acridine orange–ethidium bromide as described under Materials and Methods. Remotion of apoptotic cells was evaluated by the occurrence of macrophages engaged in the phagocytosis of apoptotic cells or debris. Monocyte/macrophage aggregates are clumps of these cells of different size: large, 450–600 mm; intermediate, 200–400 mm; small, 100–200 mm. MGC, multinucleated giant cells containing more than three nuclei englobed by the same plasma membrane. Viable lymphocytes, lymphocytes that exclude ethidium bromide. Viable apoptotic lymphocytes, lymphocytes that exclude ethidium bromide with nuclei in any of the stages of the apoptotic process. Nonviable lymphocytes, lymphocytes that do not exclude ethidium bromide. Free apoptotic bodies, nonphagocytized condensed fragments of nuclear material.

to determine culture ‘‘health’’ several factors were taken into account: (1) Removal of dead cells and debris as evaluated by a clean culture background and by evidence of phagocytosis of apoptotic material by macrophages or MGC (Figs. 1A–1E). (2) Organization of the PBMC culture, including the formation of monocyte/macrophage aggregates of increasing size (Figs. 1A–1D). (3) Formation of MGC through fusion of macrophages (Figs. 1B–1D). (4) Lymphocyte viability assessed by exclusion of ethidium bromide. (5) Lymphocyte apoptosis assessed by the presence of typical apoptotic changes in acridine orange-stained nuclei (Figs. 1A–1F). (6) Accumulation of free, nonphagocytized apoptotic bodies (Fig. 1F). According to these features, PBMC cultures were classified into types a to h. In this classification, type a and b cultures correspond to those with high phagocytic activity and maximum macrophage differentiation, while types e–h correspond to cultures with impaired

macrophage function and viability, which allows for the persistence of background dead cells and debris. Table 2 summarizes results of the observations performed after 7–15 days of culture (plateau). Organized and ‘‘healthy’’ cultures (a–c) were observed in normal controls and in patients with the less severe forms of HIV disease (Table 3), while impaired macrophage differentiation and poor phagocytic activity were observed in symptomatic HIV/ patients and in those with fullblown AIDS as exemplified in Fig. 1A. Of the HIV0 hemophilic patients, impaired macrophage phagocytosis and no MGC formation were seen only in the HTLVI/ patient. Close correlation between macrophage aggregate formation, MGC differentiation, and apoptotic cell remotion was characteristic of N-PBMC, HIV0 PBMC, and group II and III HIV/ PBMC cultures. In HIV/ PBMC cultures showing scarce macrophage aggregates and poor or absent MGC formation (e–h), nonviable lymphocytes and apoptotic debris accumulated and these facts correlated with the clinical status of the patients (Table 3) and with the AI of lymphocytes evaluated at 48 hr (16). Defective culture organization and MGC formation in group IV HIV-infected patients could not be attrib-

FIG. 2. May–Gru¨mwald–Giemsa-stained multinucleated giant cells (MGC). (A) Lymphocytes being incorporated into a MGC. (B) Macrophage linked by a narrow bridge (arrow) to the same MGC. (C) MGC containing six nuclei. The smears correspond to a 20-day culture of normal PBMC (A, B) and 15-day culture of patient 10 (group III). Original magnification, 4001.

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TABLE 3 Characteristics of PBMC Cultures from HIV/ Hemophilic Patients and Clinical Staging. PBMC Culture Patterns and Patient Distribution Frequency of distribution of different PBMC culture patterns in patients and controls Patients

a

b

c

d

e

f

g

h

Group II (3)*** Group III (12)** Group IV C2 (7)*** Group IV A (9)*** Group IV C1 (5)*** Group IV D (1) HIV0 hemophiliacs (6)* Normal donors (50)

2 2 0 0 0 0 0 0

1 3 0 0 0 0 0 4

0 6 0 0 0 0 5 40

0 1 4 3 0 0 0 6

0 0 3 4 0 0 0 0

0 0 0 2 0 0 1 0

0 0 0 0 3 0 0 0

0 0 0 0 2 1 0 0

Note. HIV/ hemophilic patients were classified according to the CDC (22) into groups II, III, IV C2, IV A, IV C1, and IV D. PBMC culture patterns (a–h) at 7–15 days were defined in Table 2. The distribution of patients from different CDC stages showing a–h PBMC culture characteristics is shown. Controls are 6 HIV0 hemophiliac patients (one of them with positive HTLV-I serology and one with positive Chagas’ disease serology) and 50 HIV0 volunteers. The number of patients studied in each group is shown in parentheses. Statistical differences (x2 test) between the distribution of patients and controls were *P õ 0.05; **P õ 0.001; ***P õ 0.00001. The percentages of monocytes in PBMC preparations put in culture at Day 0 were HIV/ hemophilic patients, group II; 13.8 { 3.1; group III: 11.5 { 1.4; group IV C2: 15.9 { 7.2; group IV A: 9.7 { 2.6; group IV C1 9.6 { 2.6; group IV D: 8.3; HIV0 hemophiliacs, 5.3 { 2.0; and in normal controls, 6.7 { 1.1.

uted to lack of monocytes in the PBMC preparations, as the proportion of monocytes at Day 0 was similar to or higher than that of controls (Table 3). DISCUSSION

The monocyte and its differentiated counterparts, macrophages and macrophage-derived polykaryons, constitute cell types that are critical for immune responsiveness. Monocytes are generated by myeloid differentiation within the bone marrow (25) and enter the circulatory system as monocytes. Complete differentiation to macrophages is associated with loss of certain surface markers, expression of other membrane molecules, and the acquisition of the adherent phenotype (26, 27). The importance of cells of the monocyte/macrophage lineage in the pathogenesis of HIV disease has been emphasized recently (28). It is thought that macrophagetropic HIV is responsible for the initial infection of the immune system and for the maintenance of HIV reservoirs. Moreover, macrophagetropic HIV appears to be crucial for the ultimate depletion of CD4 cells through infection within the lymphoid tissue (29). In this study, we examined the differentiation of monocytes in prolonged PBMC cultures from HIV-in-

fected hemophilic patients. We correlated the characteristics and organization of the cultures with the ability of phagocytic cells to remove apoptotic cells in the different stages of HIV disease. A distinct pattern of organization of the cultures and the emergence of MGC were typical of the less severe forms of HIV disease and resembled those of normal PBMC cultures. Furthermore, during the asymptomatic stages of the disease, macrophages appeared to be activated in their ability to remove and digest apoptotic cells and to generate MGC (11). MGC occur frequently in a variety of inflammatory conditions. They have been described in association with granulomatous lesions in tuberculosis, syphilis, leprosy, Crohn’s disease, rheumatoid arthritis, giant cell arteritis, and sarcoidosis (29). Fusion of monocyte-derived macrophages in different stages of maturation is thought to be involved in the formation of MGC (10). These macrophage-derived polykaryons are considered part of the normal evolution of differentiating macrophages (9, 10). Small numbers of MGC are normally formed upon in vitro culture of PBMC in the presence of normal human serum (9, 10), medium containing cytokines (30, 31), or fusion proteins (22). We observed spontaneous MGC formation in prolonged cultures of PBMC from normal individuals and HIV/ patients during the asymptomatic or less ad-

FIG. 3. Phenotype of multinucleated giant cells (MGC) and cell aggregates. Intermediate size cell clumps from a 10-day PBMC culture of patient 12 (group III) stained with fluorescent-coupled anti-HLA-DR (A) and with phycoerythrine-coupled anti-CD11c (B). MGC from an 8-day PBMC culture of patient 29 (group IV A) under phase contrast (C) or stained with CD68 and fluorescent-labeled anti-mouse IgG (D). Controls with fluorescein-labeled anti-IgG were negative. All observations were conducted at 4001.

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MACROPHAGES, MULTINUCLEATED GIANT CELLS, AND APOPTOSIS IN AIDS

vanced stages of HIV disease. These giant cells were CD68/, CD11c/, and HLA-DR/, confirming their activated macrophage phenotype (27). Moreover, in these PBMC cultures, MGC contained phagocytized apoptotic material, confirming that these cells may function like macrophages not only in host defense (9) but also in the removal of apoptotic cells. Apoptotic cell removal and degradation are thought to be important for avoiding the deleterious effects of dead cells on the surrounding tissue. However, in HIV infection, removal of infected apoptotic cells could lead to infection of the macrophages or MGC engaged in the process of clearance (18). This could be true in spite of infected host cell death through the apoptotic process, since it has been shown that unintegrated retroviral DNA may not be destroyed and infectivity can be retained (33). In fact, a pathway for the recycling of HIV genetic information after the death of the initial host cell (DNA-mediated phagoinfection) has been recently proposed as an alternative pathway of HIV infection (18–19). This could be important in the early stages of HIV infection, when macrophage function is still preserved, allowing the efficient ingestion of apoptotic cells containing HIV DNA, and when lymphokine-induced macrophage activation can enhance macrophagetropic HIV growth (34). Furthermore, through their attachment and intimate contact with macrophages in the cellular clumps, noninfected apoptotic HIV/ lymphocytes (35) could favor macrophage infection. In this study we have shown that long-term cultures of PBMC from infected individuals in the absence of exogenous stimulation (allogeneic cells, T cell mitogens, or IL-2 stimulation) (34, 13) can be obtained. Activated clearance of apoptotic cells and enhanced differentiation of macrophages into MGC appeared to be the consequences of in vivo activation of macrophage function in HIV/ patients during the asymptomatic or less advanced stages (a–b PBMC culture morphology, Tables 2 and 3). Thus, PBMC cultures from group II and III patients revealed greater clearance activity than those of normal controls. In the more advanced stages, macrophage function and differentiation deteriorated, perhaps in relation to the depletion of the lymphocytes that provide the necessary cytokines for macrophage differentiation (36). Therefore, this simple experimental model may be of benefit to examine the cellular milieu in which HIV replicates in an individual host in relation to pathogenesis as well as for the assay of antiretroviral drugs. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Ms. Marta Felippo and Graciela Mendez, the generous cooperation of Drs. R. Perez Bianco, M. Tezanos Pinto, M. Candela, M. Cermelj,

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and M. Narbaitz, the hemotherapy personnel of IIHEMA, Dr. G. Fernandez-Alonso of CEO-Academy of Medicine, and Ms. P. and D. Careri from the Argentine Foundation of Hemophilia. This work was performed with funds of CONICET (PID 3348), Fundacion Roemmers, and Fundacion Mosoteguy (Argentina). REFERENCES 1. Ameisen, J. C., and Capron, A., Cell dysfunction and depletion in AIDS: The programmed cell death hypothesis. Immunol. Today 12, 102–105, 1991. 2. Gougeon, M. L., and Montagnier, L., Apoptosis in AIDS. Science 260, 1269–1270, 1993. 3. Gerschenson, L. E., and Rotello, R. J. Apoptosis: A different type of cell death. FASEB J. 6, 2450–2455, 1992. 4. Martin, S. J., Green, D. R., and Cotter, T. G., Dicing with death: Dissecting the components of the apoptosis machinery. Trends Biochem. Sci. 19, 26–30, 1994. 5. Cohen, J. J., Overview: Mechanisms of apoptosis. Immunol. Today 14, 126–130, 1993. 6. Thompson, C. B., Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462, 1995. 7. Bursch, W., Oberhammer, F., and Schulte-Hermann, R., Cell death by apoptosis and its protective role against disease. Trends Pharmacol. Sci. 13, 245–251, 1992. 8. Savill, J., Fadok, V., Henson, P., and Haslett, C., Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14, 131– 136, 1993. 9. Schlesinger, L., Musson, R. A., and Johnston, R. B., Functional and biochemical studies of multinucleated giant cells derived from the culture of human monocytes. J. Exp. Med. 159, 1289– 1294, 1984. 10. Mariano, M., and Spector, W. G., The formation and properties of macrophage polykaryons (inflammatory giant cells). J. Pathol. 113, 1–19, 1973. 11. Hassan, N. F., Kamani, N., Meszaros, M. M., and Douglas, S. D. Induction of multinucleated giant cell formation from human blood-derived monocytes by phorbol myristate acetate in in vitro culture. J. Immunol. 143, 2179–2184, 1989. 12. Black, M. M., and Epstein, W. L., Formation of giant cells in organized epithelioid cell granulomas. Am. J. Pathol. 74, 263– 274, 1974. 13. Pantaleo, G., and Fauci, A. S., Apoptosis in HIV infection. Nature Med. 1, 118–134, 1995. 14. Lewis, D. E., Ng Tang, D. S., Adu-Oppong, A., Schober, W., and Rodgers, J. R., Anergy and apoptosis in CD8/ T cells from HIVinfected persons. J. Immunol. 153, 412–420, 1994. 15. Gougeon, M. L., and Montagnier, L., New concepts in the mechanisms of CD4/ depletion in AIDS, and the influence of opportunistic infections. Res. Microbiol. 143, 362–368, 1992. 16. Ruibal-Ares, B., Riera, N. E., Felippo, M., Vernava, D., PerezBianco, R., and de Bracco, M. M. E. Apoptosis in HIV-infected hemophilic patients. Immunol. Infect. Dis. 5, 159–166, 1995. 17. Duke, R. C., and Cohen, J. J., Morphological and biochemical assays of apoptosis. In ‘‘Current Protocols in Immunology’’ (J. E. Coligan, A. M. Kruisbek, D. H., Margulies, E., Shevach, and W. Strober, Eds.), pp. 3.17.1–3.17.7, Wiley, New York, 1991. 18. Kornbluth, R. S., The immunological potential of apoptotic debris produced by tumor cells and during HIV infection. Immunol. Lett. 43, 125–132, 1994. 19. Kornbluth, R. S., Significance of T cell apoptosis for macrophages in HIV infection. J. Leukocyte Biol. 56, 247–256, 1994.

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Received March 7, 1996; accepted with revision August 23, 1996

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