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In vivo activation of alveolar macrophages in ovine lentivirus infection
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
In Vivo Activation
55,
355-367 (1990)
of Alveolar Macrophages Lentivirus Infection
in Ovine
GENEVI~E CORDIER,* GRBGOIRECozoN,*-t TIMOTHY GRmNLmD,*~t FRANCOIS ROCHER,t FRANCOISGUIGUEN,~ SYLVIANE GUERRET,~ JEANBRUNE,~ANDJEAN-FRANCOISMORNEX~ *INSERM V 80, CNRS VRA 1177. Vniversitk Claude Bernard (Centre de Cytojluoromktrie), H6pital Edouard Herriot, Lyon, France; TLaboratoire d’lmmunologie et de Biologic Pulmonaire, Service de Pneumologie, Hbpital Louis Pradel et Facultk de MPdecine Grange Blanche, Lyon, France; SLaboratoire Associd de Pathologie des Petits Ruminants, INRA, Ecole Nationale VSrinaire, Marcy I’Etoile, France; and flnstitut Pasteur, CNRS VRA 167, Lyon, France
Sheep infected by visna-maedi virus, a lentivirus related to the human immunodeficiency virus, develop a chronic interstitial lung disease. Since monocyte/macrophages are known to be specifically infected by visna-maedi virus, we investigated the role of macrophages in the appearance of pulmonary lesions in animals with naturally occurring disease. Alveolitis in maedi leads to a doubling in bronchoalveolar lavage total cell counts and of macrophages as compared to normal sheep. A significant increase in the relative percentage of neutrophils was also observed, accompanied by an increased spontaneous release of neutrophil chemotactic activity by alveolar macrophages of diseased animals, suggesting that they may be activated. Macrophage activation is also demonstrated by the observation of a significant (X 3) increase of spontaneous fibronectin release by alveolar macrophages from maedi lungs, and furthermore by the high level expression of major histocompatibility complex class II antigens on most of these celfs. Thus viral infection, although restricted to a small population of macrophages, is able to modulate extensive activation of macrophages in the lung. Activated macrophages release mediators likely to play a role in the development of the alveolitis and the parenchymal desorganization. These findings may be relevant to our understanding of the mechanisms by which human immunodeficiency virus infection leads to pulmonary disease other than that caused by opportunistic infections. 0 1990 Academic Press, Inc.
INTRODUCTION
Visna-maedi virus, isolated from sheep, is a member of the lentiviruses, a subfamily of nononcogenic retroviruses (1, 2). Lentiviruses are known to infect various species: goats (caprine arthritis encephalitis virus, CAEV); horses (equine infectious anemia virus); cattle (bovine immunodeticiency virus); cats (feline immunodeficiency virus, FIV); and primates (human immunodeficiency viruses, HIV-l and HIV-2 in humans and simian immunodeficiency viruses, SIV, in monkeys), While some of these viruses, e.g., HIV-I, SIV, and FIV, are associated with an immunodeficiency syndrome, all of them induce chronic and/or degenerative diseases affecting the lungs, joints, mammary glands, and the central nervous system (1, 2). In sheep, spontaneously or experimentally infected by visna-maedi virus, the primary target organ is the lung where an interstitial lung disease known as ovine progressive pneumonia or maedi develops (3-5). Lung pathology includes chronic interstitial inflammation with dense cellular infiltration, hyperplasia of smooth 355 0090- 1229FM $1.50 Copyright All rights
Q 1990 by Academic Press, Inc. of reproduction in any form reserved.
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muscle cells in the alveolar septa, slight fibrosis, peribronchial and perivascuiar lymphoid hyperplasia, and epithelial proliferation in small bronchi and bronchioles (3, 4) and (R. Loire, unpublished observations). An interstitial lung disease with similar pathological features has also been reported in infections by CAEV (6) and HIV-l (7, 8). Most lentiviruses infect cells of the monocyteimacrophage lineage in iai~j (reviewed in Refs. (1, 2)). The effect of this infection on mononuclear phagocyte functions remains controversial; decreased phagocytic (9). chemotactic (lo), and chemiluminescent (11) responses of blood monocytes have been reported in lwmans in the acquired immunodeficiency syndrome, whereas increased secretion of interleukin-1 (IL-l) by monocytes could be detected as a consequence of infection by HIV-l (12, 13) or in the presence of its products (14). Since macrophages can exert multiple functions and can modulate inflammatory processes (reviewed in Ref. (15)). some of the pathologic events observed in lentiviral in fections may be due to these cells. The interstitial lung disease due to visna-maedi virus allows the assessment of the in viva macrophage functions in the course of natural infection in the absence of opportunistic infections. By analyzing cells collected by bronchoalveolar lavage from lungs of maedi animals, we could demonstrate macrophage activation and the release of mediators involved in the pathogenesis of the interstitial lung disease. MATERIALS
AND
METHODS
Sheep Lungs
Adult sheep lungs were obtained from slaughterhouses. Lungs with macroscopic or histological evidence of parasitic infections were excluded, as were those showing hemorragic and bacterial contamination after adequate examination of bronchoalveolar lavage fluid. The diagnosis of maedi (n = 31) was based upon histological examination. Diagnostic criteria included peribronchovascular lymphoid nodules, alveolitis, and increased number of smooth muscle cells and fibroblasts (3). Control lungs (rz = 26) were defined by the absence of macroscopic and microscopic abnormal features. Bronchoalveolar Lavuge
In order to collect alveolar cells, a flexible canula was passed through the trachea and wedged into one of the main stem bronchi. Three hundred milliliters of Hank’s balanced salt solution (HBSS; BioMerieux, Charbonnieres les Bains, France) was infused and aspirated after gentle massage of the lung. The recovered liquid was immediately filtered through sterile gauze. Total cell counts were established from numeration with a hemocytometer. Differential counts of bronchoalveolar lavage cells were performed on cytocentrifuge (Cytospin, Shandon S.A., Enguy, France) smears stained using ’ ‘Diff-Quick” (Merz & Dade AG, Dtidingen, Switzerland). Cell Suspensions
Cells were separated from bronchoalveolar
lavage fluid by centrifugation
for 15
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min at 400g. Bronchoalveolar cells were washed twice in HBSS and resuspended at lo7 cells/ml in culture medium (RPM1 1640 medium supplemented with a mixture of penicillin, 100 U/ml; streptomycin, 50 t&ml; and vancomycin, 20 pg/ml). Cell viability was measured at this step by trypan blue exclusion and was consistantly over 85%. Evaluation of Neutrophil Chemotactic Activity (NCA) Generated by Alveolar Cells For evaluating the generation of NCA by alveolar macrophages, lo6 cells/ml RPM1 1640 medium without antibiotics were incubated in sterile tissue culture flasks for 3 hr in 5% CO2 in air, at 100% humidity. Supernatants were harvested by aspiration and freed of cells or debris by centrifugation at 28,000g for 10 min; they were stored at -20°C before testing. Neutrophil chemotactic activity in these supernatants was demonstrated by measuring the migration of human neutrophils in a 48well microchemotaxis chamber (Neuroprobe, Cabin John, MD) through a 3-pm micropore filter (PVPfree filter, Nucleopore, Pleasanton, CA). Human neutrophils were prepared from peripheral blood of healthy volunteers by gradient centrifugation on Mono-poly resolving medium (Flow Laboratories, Les Ulis, France) according to the supplier’s methodology. The resulting neutrophil suspensions contained more than 98% neutrophils. Aliquots (25 ~1) of supernatants to be assayed were placed in the lower wells, the filter sheet was fixed in place to separate bottom from top wells, and cells were added to the upper wells (3 x 106/ml culture medium, 40 t.~l). The chamber was incubated for 45 min at 37°C. During incubation, migrating cells penetrated the filter and remained attached to its lower surface. At the end of the incubation the filter was removed and cells which had not migrated into the pores were removed by drawing it over a wiper blade. The filter was then fixed and stained with Diff-Quick stain. Chemotactic activity is usually expressed as the number of cells counted per high power field of the stained filter. In preliminary assays we observed that the staining could be evaluated optically. Preliminary experiments showed that the light absorbance of the fixed stained cells, integrated with a spectrophotometer (Vernon PHI 6, Paris, France) over each spot corresponding to a well, increased regularly with the number of cells present over the range of 0 to 150 x lo3 under experimental conditions (data not shown). We thus used light absorbance as an index of migration. The results were standardized between assays by defining 100 migration units as the maximal light absorbance obtained using formylL-methionyl-L-leucyl-L-phenylalanine oligopeptide (Calbiochem, FranceBiochem, Meudon, France) at lop7 M as an attractant (optimal concentration under our experimental conditions). The relative attraction of culture supernatants was expressed as units per milliliter corresponding to the activity released by lo6 macrophages per milliliter. Fibronectin Release by Alveolar Macrophages The release of fibronectin by alveolar cells was measured in culture supematants. Based on the kinetics of production observed by Rennard et al. (16) for
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human alveolar macrophages, 5 x lo6 cells in 5 ml culture medium were incubated for 21 hr at 37°C in 5% CO2 in air at 100% humidity. In some experiments, alveolar cells from healthy lungs were cultured in the presence of lipopolysaccharide (LPS; Difco laboratories, Detroit, MI) at 10 l&ml or freshly opsonized zymosan particles (Sigma, St. Louis, MO) at 5 x lo6 particles/ml. Cell-free supernatants obtained by centrifugation (28,OOOg, 10 min) were stored frozen at - 20°C until used. The fibronectin released in supernatants was evaluated using a sandwich ELISA relying on the affinity of fibronectin for gelatin on the one hand and the binding of anti-tibronectin antibodies on the other. Purified rabbit anti-human fibronectin antibodies were prepared at the Institut Pasteur de Lyon (17). Crossreactivity with purified sheep fibronectin was demonstrated by Ouchterlony immunoprecipitation; in addition, no reactivity was demonstrated toward fibronectin-depleted sheep serum after a two-step affinity-chromatography purification (data not shown). The microplate (Nunc-Immuno, Roskilde, Denmark) was coated with a gelatin solution prepared by warming 2.5 mg/ml of gelatin (Bio-Rad Laboratories, Richmond, CA) in carbonate-bicarbonate buffer (Na,CO,, 15 n&f; NaHCO,, 35 mM; pH 9.3) for 10 min at 90°C. Two hundred microliters of this solution was delivered per well and incubated for 18 hr at room temperature. After five washings with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma) (PBS-Tween), each well received 200 ~1 of alveolar cell supernatant for a further incubation of 2 hr at room temperature. Then, the supernatant was discarded and the wells were again washed five times with PBS-Tween. To each well was then added 200 l.~l of a 1:250 dilution of the anti-fibronectin antibodies. After incubation for 1 hr and washing (~5) with PBS-Tween, 200 ~1 of peroxidaseconjugated goat anti-rabbit Ig antibodies (Biosys, Compibgne, France) at optimal dilution (1:4000) in the presence of 0.1% goat serum (Seralab, Sussex, England) was added for 1 hr. The excess reagent was discarded and the plates were washed five times with PBS-Tween. Finally the plates were incubated for 15 min with 200 p.l/well of substrate O-phenylenediamine dihydrochloride (Sigma). The enzymatic reaction was stopped by adding 100 pi/well of H,SO, (2 N) and the absorbance at 492-650 nm was read in an ELISA processor (SLT210, Kontron AG, Zurich, Switzerland). The standard curve for dosage in each test was obtained using serial dilutions (1:2000 to l:lOO,OOO) of a pooled normal sheep sera preparation stored frozen at - 20°C until use. This preparation contains 298 &ml of fibronectin as assessed by Dr. S. Bozena-Begin (University of Sherbrooke, Sherbrooke, Canada). Amounts of fibronectin in the test supernatants were evaluated from the mean value of triplicate measures. Results are expressed as nanograms per milliliter of tibronectin released by lo6 macrophages per 24 hr. Macrophage
Membrane
Antigen
Expression
Indirect immunofluorescence was used to determine membrane antigenic expression by alveolar cells. Sheep major histocompatibility complex (MHC) class II antigens were identified using two specific monoclonal antibodies: SBU-II (clone 28-l), a mouse IgGl anti monomorphic MHC class II antigen (18) supplied
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by Dr. Brandon (University of Melbourne, Australia), and SW (clone 73.2), a rat Ig anti-monomorphic MHC class II antigen (19) kindly donated by Dr. Myasaka (Base1 Institute for Immunology, Switzerland). In addition, hybridoma 175, a mouse IgM K, anti-sheep myeloid and erythroid cells (19), was used to identify alveolar cells. A purified preparation of FITC-conjugated goat IgG anti-mouse IgG + IgM (FITC-GAMIG; Biosoft, Paris, France) was used as the second step reagent. Samples of lo6 cells were incubated for 30 min in 50 ~1 primary antibody preparation at the optimal dilution. They were then washed twice with PBS containing 15 mM NaN, plus 1% BSA (PBS-BSA-Azide) and the pellets were resuspended in 50 ~1 of the appropriate dilution of FITC-GAMIG for a further 30 min. Cells were washed twice and fixed in 700 ~1 of PBS-BSA-Azide with 1% formaldehyde. Control samples incubated with FITC-GAMIG alone were included in each series. Cells were analyzed in a Cytofluorograf 50 H (Ortho Instruments, Westwood, MA) equipped with a 5 W Argon-ion laser and interfaced with an interactive computer developed in this laboratory (20). The 488-pm laser line was used at 400 mW for excitation. Three parameters were recorded for each cell: the forward angle light scattering (FAS) which is related to cell diameter, the right angle light scattering (RAS) which gives information on the internal structural properties of the cell, and green fluorescence related to the fixation of antibodies. Preliminary experiments showed that using a log scale on both FAS and RAS parameters it was possible to discriminate between various clusters. Analysis of the correlated fluorescence for each cluster in sample aliquot stained with 175 allowed the identification of macrophages (175-positive, high FAS and RAS). In contrast, lymphocytes were shown to be 175-negative cells with low FAS and RAS whereas neutrophils were characterized as 175-positive cells with intermediate FAS and RAS. Fluorescence histograms were recorded using the gating on FAS and RAS parameters. For each sample, 10,000 to 20,000 cells were accumulated in order to get a fluorescence histogram with at least 5000 cells within each gate. The percentage of positive cells was calculated for biphasic distribution by counting cells above a threshold established on control cells. When the test and control histograms overlapped, the threshold was set at the channel above the overlapping area. Fluorescence intensity was expressed as arbitrary units. Evidence of Viral Infection The presence of lentivirus in alveolar macrophage specimens was demonstrated by the occurrence of a cytopathic effect following cocultivation with permissive cells. Briefly, bronchoalveolar lavage cells were resuspended in culture medium at 2-3 x 106/ml, then l-2 x 106/ml of freshly trypsinized ovine skin fibroblasts (ID05 strain) were added and the coculture was incubated in 25-cm* Falcon flasks. Cultures were examined every day by phase contrast for evidence of cytopathic effect. Viral infection was demonstrated by the appearance by Days 10-30 of culture of syncytia (>g nuclei/cell) observed following fixation and staining of the fibroblasts with May-Grumwald-Giemsa. Negative cultures were monitored for over 60 days.
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Statistical Analysis Results are presented as means t SEM. Statistical Student’s two-tailed test.
analysis was made using
RESULTS
Accumulation
of Inflammatory
Cells within the Alveolar
Spaces
Maedi is characterized by an alveolitis. This was demonstrated by direct sampling of alveolar cells using bronchoalveolar lavage. Both total and differential cell counts were evaluated (Table I), the number of alveolar cells is significantly increased in sheep with maedi (n = 12) when compared to healthy controls (n = 15). In lavages from normal lungs (n = 19). alveolar cells were mainly macrophages, the distribution was modified in lavages from maedi lungs (n = 20) with a significant increase in the percentage of neutrophils (Table 1). Although the percentage of macrophages was decreased (Table l), the absolute macrophage counts were significantly increased from 0.9 x 106/ml 2 0.1 in healthy lungs to 1.8 x 106/ml + 0.5 (P < 0.05) in the maedi ones. There was no significant change of the lymphocyte percentage (Table 1). Generation
of NCA by Alveolar
Macrophages
To investigate a possible cause for the increased neutrophil content within the alveolar spaces, we evaluated the generation of NCA by alveolar macrophages. The chemotactic activity of macrophages culture supernatants was assessed using human neutrophils as indicator cells. To ensure that our culture conditions allowed the generation of NCA and that human neutrophils could react to it, we tested supernatants from normal sheep alveolar cells stimulated with zymosan particles and LPS. These supernatants repeatedly induced migration of human neutrophils as shown by the migration values observed: 49.5 -C 2.0, 38.2 + 6.5, and 14.3 k 3.8 units/ml, respectively, for zymosan. LPS, and control medium in seven separate experiments (Fig. 1). Supernatants of bronchoalveolar lavage cells from maedi lungs (n = 18) cultured in the absence of stimulants were compared to those obtained from healthy lungs (n = 15). The results (Fig. 1) indicate that alveolar cells from infected animals spontaneously produce increased amounts of TABLE CELLULARCOMPOSITION
Normal Maedi
I
OF BRONCHOALVEOLAR
Cell counts” (lo6/ml)
Macrophage@
1.7 k 0.3 (0.6-5.4) 3.7 L 1.1 (0X-12.0)
78.0 + 2.0 62.0 r 4.0
5.0 t 1.0 13.0 t 3.0
P < 0.05
P < 0.05
P < 0.05
u Means 2 SEM (range). b Expressed as percentage of total cells. ’ Expressed as percentage of macrophages. ’ Not significant.
Neutrophils”
LAVAGE
Lymphocytes” 15.0 t 2.0 18.0 -t 3.0 nsJ
MHC clas:, II positive macrophages”,’ IS.5 _t 4.0 (U-37) SO.1 + 4.5 (24-86)
P i 0.05
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60 1 50-
40-
30-
20-
lo-
O-
normal
maedi
IlOriIXil
+LPS
normal +Zymosan
FIG. 1. Generation of NCA by alveolar macrophages from healthy (n = 19) and maedi (n = 18) lungs. Supematants were prepared from lo6 cells/ml incubated for 3 hr in culture medium. Migration indexes were measured as described under Materials and Methods. Bars indicate the mean value (2 1 SEM) of migration index observed for each group.
NCA (28.1 ? 4.7 units/ml vs 16.6 + 3.1 units/ml, P < 0.05) giving one possible explanation for the increased proportion of neutrophils in the alveolar spaces. Fibronectin Release by Alveolar Macrophages
Fibronectin is a mediator which could modulate some of the lung parenchymal lesions; we thus evaluated the spontaneous release of fibronectin by alveolar macrophages. Fibronectin was measured in culture supernatants of alveolar cells from maedi lungs (n = 11) or from normal lungs (n = 14), with or without LPS or zymosan stimulation (Fig. 2). It can be seen that the maedi bronchoalveolar lavage macrophages spontaneously released three times as much libronectin as did the normal cells (11.4 + 3.2 r&ml vs 3.5 ? 0.9 rig/ml per 24 hr, P < 0.05). Interestingly enough, unlike the situation for NCA, stimulation of normal macrophages did not increase their production of libronectin (Fig. 2). This suggests that different mechanisms are involved in the control of the production of these two factors. MHC Class II Antigen Expression by Alveolar Macrophages
Although MHC Class II antigens are constitutively expressed by macrophages, their level of expression is tightly regulated. In suspensions prepared from normal lungs (n = 15) the percentage of MHC class II antigen-positive macrophages was low (15.5 + 4.0, Table 1) using either the SW mAb or the SBU-II mAb. both specific for a monomorphic epitope of sheep MHC class II antigens. In suspensions prepared from maedi lungs (n = 13), we observed an increase in the percentage of labeled cells (50.1 2 4.5%; P < 0.05 when compared to healthy specimens). A change in antigen density could also be observed as depicted by the shift of the fluorescence profile (Fig. 3).
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maedi
normal +I,PS
normal +zymosan
FIG. 2. Fibronectin release by alveolar macrophages. Fibronectin was assayed by ELlSA as described under Materials and Methods. Supernatants were prepared by culture of 5 x IO’ cells in 5 ml culture medium for 21 hr from maedi (n = 12) and healthy lungs without (n = 15) and with stimulation by LPS (50 &ml, n = 13) and zymosan particles (5 x lO?ml. n = 8). Bars indicate the mean value (21 SEM) of tibronectin (r&ml) released per IO’ cells/24 hr for each group.
Evidence of Lentiviral Infection Alveolar macrophages were cultured with ovine tibroblasts in order to detect lentiviral infection. No cytopathic effect was observed when fibroblasts were cultured alone. Similarly, macrophages from 19 microscopically normal lungs were cultured with fibroblasts without evidence of syncitia for up to 2 months (three to four passages). In contrast, macrophages from 11 out of 12 maedi lungs led to syncitia formation. Further characterization of these isolates included the demonstration of a reverse transcriptase activity (two cases, data not shown) and the presence of viral particles by electron microscopy examination (four cases, data not shown). Thus alveolar macrophages collected from maedi lungs harbored a lentivirus. DISCUSSION
Lentiviruses induce chronic inflammatory and/or degenerative disorders within multiple organs. In sheep infected by visna-maedi virus the lung is the main target organ. Most of the attention has so far focused on the slow development of the disease and its delay in appearance. Although the virus is known to infect the cells of the monocyte/macrophage lineage specifically, no s:tndies have investigated the effect of lentiviral infection on macrophage functions. In this study, using bronchoalveolar lavage to gain access to the inflammatory cells at the site of the disease, we can characterize the alveolitis process occurring in spontaneously infected animals. We also evaluate the ability of alveolar macrophages to produce mediators involved in the attraction and the proliferation of the various cell types observed in the affected lung. Distribution of alveolar cells in healthy lungs is similar to that previously described (21). In maedi lungs, diagnosed on pathological grounds as described
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Maedi
fluorescence intensity FIG. 3. Fluorescence profiles of alveolar macrophages. Cells prepared from healthy (left) and maedi (right) lungs were stained for MHC class II antigens using the SBU-II mAb (A) and for a myeloid antigen using the 175 mAb (B). Histograms were obtained from at least 5 x lo3 macrophages gated according to their RAWFAS characteristics. The change in antigen density is demonstrated by the shift of the histograms.
elsewhere (3) besides the pathological evidence of alveolar wall inflammation there is a luminal alveolitis as shown by the significant increase in the number of alveolar cells. Cells comprising this alveolitis are mainly macrophages and neutrophils without significant increase in the percentage of lymphocytes. The increased macrophage content in the alveoli and the dense infiltration by neutrophils are known from histological studies in animals with spontaneous advanced maedi (3). As has previously been shown in the human (22), cells collected by bronchoalveolar lavage reflect the inflammatory process occurring in the lung and we therefore used bronchoalveolar lavage cells for the assessment of the macrophage function in the lung. Evidence of Macrophage Activation
In agreement with the observed neutrophilic alveolitis we find an increased spontaneous NCA release by alveolar macrophages from maedi lungs. It is observed in the absence of exogenous stimulatory agents. A variety of agents, including microorganisms, noninfectious particles, and immune complexes, are able to induce the generation of NCA (23). A role for immune complexes remains a distinct possibility since viral antigen production continues in antibody positive
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sheep, and production of IgGl in synovia of CAEV-infected goats probably represents a local response (24, 25). No antibodies to visna-maedi virus were found in one study of bronchoalveolar lavage liquid of maedi sheep (26), suggesting a low probability of local complex formation; however, our recent observations suggest that anti-viral antibodies may sometimes be found in bronchoalveolar lavage fluids (D. Ecochard, unpublished data). One neutrophil chemotactic factor has been shown to be delicately regulated. This recently described monocytederived neutrophil chemotactic factor is induced by II- 1 and tumor necrosis factor but not by interferon-y (IFN-y) (27). This would suggest complex interactions between infected and noninfected cells at the site of the disease. Fibronectin is able to stimulate the growth of fibroblasts thus playing a role in modulating damage and repair in inflammatory processes (16, 22, 28). We demonstrate an increased spontaneous release of fibronectin by alveolar macrophages from maedi lungs. Fibronectin is released in small amounts by alveolar macrophages from normal sheep lungs, LPS and immune complexes were unable to increase fibronectin secretion as previously shown in humans (28). Fibronectin production is regulated by steady-state levels of mRNA; currently no information is available concerning the controlling elements for fibronectin gene expression or the mechanism modulating the fibronectin mRNA life span. In maedi lungs most of the alveolar macrophages express high levels of MHC class II antigens. A small percentage of alveolar macrophages from normal iungs express these antigens in agreement with prevous observations (7). Modulation of class II antigen expression has been largely documented among changes occurring in activated macrophages (15). Increased expression of MHC class II antigens by macrophages of maedi animals together with the above observations, i.e., increased release of NCA and fibronectin, support the existence of an activation process within the lung in maedi animals. The mediators released in situ by activated alveolar macrophages are likely to play a role in the development of the lung pathologic changes as shown in human interstitial lung diseases (16, 22, 23, 29). Mechanisms
of Macrophage
Activation
The mechanisms leading from macrophage infection to macrophage activation remain to be elucidated. In this study, alveolar macrophages from animals with histologically proven maedi harbor a visna-maedi-like virus and normals do not. A direct activation of macrophages by visna-maedi virus could therefore be possible. Two lines of evidence argue against this: (i) whereas in our study up to 50% of macrophages are activated as assessed by their increased expression of MHC class II antigens, only 0.1 to 1% macrophages are known to express virus in infected animals (reviewed in Ref. (1)) and (ii) in vitro infection of macrophages does not lead to an increased expression of MHC class II antigens (30). An indirect mechanism of activation is more probable. Macrophage activation is under the control of signals operationally defined as macrophage-activating factors. Most of them are released by activated lymphocytes. One of the most potent in vivo and in vitro is IFN-y (31) which is well known as an inducer of Ia antigen expression (31, 32) and is able to enhance the secretion of various proteins including, in the mouse, the production of tibronectin (33). Such a cellular interac-
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tion is likely to occur since (i) an interferon is spontaneously released by lymphocytes cocultured with in vitro-infected macrophages (34) and by lymphocytes collected from lymph nodes of experimentally infected lambs (39, moreover it is present in synovial fluid from naturally infected sheep (36) and (ii) we have demonstrated activated alveolar T lymphocytes in maedi lungs (37). In conclusion, the interstitial lung disease observed in spontaneous visna-maedi virus infection is mediated by macrophage activation. Activated macrophages show increased production of mediators able to modulate the interstitial lung disease in situ. Macrophages are likely to be activated through complex cellular interactions as a consequence of viral infection. Since the genomic organization and the basic biological features of the host-virus interactions are remarkably similar for visna-maedi virus and HIV-I, the interstitial lung disease due to visnamaedi virus offers a useful model for the study of lentiviral-induced inflammatory disease in humans. ACKNOWLEDGMENTS The authors thank Mr. Roussin and Mr. Lefevre for providing them with animal lungs; Dr. M. Fontaine for virus isolation; Dr. R. Loire for performing the pathological examinations; Dr. S. BozenaBegin and Dr. A. Cantin for tibronectin assay in the reference preparation; Dr. G. Caillot for the preparation of purified sheep tibronectin and Bbronectin-depleted sheep serum; Dr. M. Myasaka for the generous gift of SW and 175 mAb; and Mss. V. Pelamatti and C. Quintin for expert secretarial assistance. They are especially grateful to Professor J. P. Revillard for advice and valuable discussion during this work. This work was supported in part by grants from: INSERM (CRE 865011); Minis&e de la Recherche et de I’Enseignement Superieur (Programme National de Recherche sur le SIDA); Minis&e de la Recherche et de la Technologie (FRT); Universite Claude Bernard, Departement de Biologie Humaine et Fact&C Grange Blanche; Fond Special des Comites Departementaux de Lutte contre les Maladies Respiratoires (87/MR21, 88 MR/13); Hospices civils de Lyon; Institut Mtrieux, Marcy I’Etoile (France).
REFERENCES 1. Haase. A. T., Pathogenesis of lentivirus infections. Nature (London) 322, 130-136, 1986. 2. Narayan, 0.. and Clements, J. E., Biology and pathogenesis of lentiviruses. J. Gen. Viral. 70, 1617-1639. 1989. 3. Georgsson, G., and Palsson, P. A., The histopathology of Maedi: A slow viral pneumonia of sheep. Vet. Pathol. 8, 63-89, 1971. 4. Lairmore, M. D., Rosadio, R. H., and De Martini, J. C., Ovine lentivirus lymphoid interstitial pneumonia: Rapid induction in neonatal lambs. Amer. J. Pathol. 125, 173-181, 1986. 5. Momex, J. F., Greenland, T., Cordier, G., Rocher, F., Cozon, G., Loire, R., Patet, J., Guiguen, F., Cadore, J. L., and Brune, J., Diffuse interstitial lung disease in sheep caused by Maedi/Visna virus. In “The Alveolar Macrophage” (C. Sorg, Ed.), pp. 96100. Regensbers and Bierman, Munster. 1988. 6. Straub. 0. C., Caprine arthritis encephalitis: A model for AIDS? Intervirology 30, 45-50. 1989. 7. Marchevsky, A., Rosen, M., Crystal, R., and Kleinerman, J., Pulmonary complications of the acquired immunodeficiency syndrome: A clinicopathologic study of 70 cases. Hum. Pathol. 16, 659-670, 1985. 8. Joshi, J., Oleske, J., Minnefor, B., Saad. S., Klein. K., Singh, R., Zabala. M., Dadzie, C., Simpser, M., and Rapkin, R., Pathologic pulmonary findings in children with the acquired immunodeficiency syndrome: A study of ten cases. Hum. Pathol. 16, 241-246, 1985. 9. Estevez, M. E., Ballard, I. G., Diez, R. A., Planes, N., Scaglione, C., and Sin. L., Early defect of phagocytic cell function in subjects at risk for acquired immunodeficiency syndrome. &and. J. Immunol. 24, 215-221, 1986.
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AL.
10. Smith, P. D., Chura, K., Masur, H., Lane, H. C., Fauci, A. S., and Wahl, S. M.. Monocyte function in the acquired immunodeficiency syndrome defective chemotaxis. J. C/in. invest. 74, 2121-2124, 1984. Il. Braun. D. P., Kessler, H., Falk, L., Paul, D.. Harris, J. E., Blaauw, B., and Landay A., Monocyte functional studies in asymptomatic, human immunodeficiency disease virus (HIV)-infected individuals. 1. C/in. Immunol. 8, 486-494, 1988. 12. Roux-Lombard, P., Modoux, C., Cruchaud, A., and Dayer, J. M.. Purified blood monocytes from HIV-l-infected patients produce high levels of TNF u and IL-I. C/in. Immunol. Immunopnthol. 50, 374-384, 1989. 13. Folks, T. M.. Justement, J., Kinter, A., Dinarello, C. A., and Fauci A. S.. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238, 800-802, 1987. 14. Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Hare]-Bellan, A.. and Farrar. W. L,.. Human immunodeticiency virus glycoprotein (gp 120) induction of monocyte archidonic acid metabolites and interleukin 1. hoc. Nat/. Acad. Sci. USA 86, 621-625. 1989. 15. Adams, D. O., and Hamilton, T. A., The cell biology of macrophage activation. Anna, Ret. lmmunol.
2, 283-313,
1984.
16. Rennard, S. I., Hunninghake, G. W., Bitterman, P. B.. and Crystal, R. G., Production ot fibronectin by the human alveolar macrophage: Mechanism for the recruitment of fibroblasts to sites of tissues injury in interstitial lung diseases. Proc. Nat/. Acad. Sci. USA 78,7147-7151, 1981. 17. Grimaud, J. A.. Druguet M.. Peyrol, S., and Guerret. S., Collagen. In “Methods of Enzymatic Analysis, Proteins and Peptides” (H. U. Bergmeyer, Ed.), Vol. 9. pp. 186195, VCH Publishers, Weinheim, 1986. 18. Puri, N. K., Mackay, C. R., and Brandon, M. R., Sheep lymphocyte antigens (OLA). Il. MaJor histocompatibility complex class II molecules. Immuno/ogy 56, 725-734, 1985. 19. Miyasaka, M., McClure, S., Hein, W. R., and Tmka, Z., Differentiation antigens in lymphohemopoietic system of the sheep. In “Differentiation Antigens in Lymphohemopoietic Tissues” (M. Miyasaka and 2. Tmka, Eds.), pp. 499-523, Dekker, New York/Base]. 1988. 20. Momex, J. F., Cordier, G.. Pages, J., Lefebvre. R., Revillard, J. P.. Vergnon. J. M.. Vincent. M., Cordier, J. F., and Brune, J., Pulmonary sarcoidosis: Flow cytometry measurement of lung T cell activation. J. Lab. Clin. Med. 105, 70-76, 1985. 21. Rola-Pleszcynski. M., Sirois, P., and Begin, R., Cellular and humoral components of bronchoalveolar lavage in the sheep. tung 159, 91-99, 1981. 22. Keogh. B. A., and Crystal. R. G., Alveolitis: The key to the interstitial lung disorders. %NU~ 37, I-10. 1982. 23. Hunninghake, G. W., Gadek, J. E., Fales, H. M., and Crystal. R. G.. Human alveolar macrophage-derived chemotactic factor for neutrophils. Stimuli and partial characterization. J. C/in. Invest. 66, 473-483, 1980. 24. Johnson, G. C.. Barbet, A. F., Klevjer-Anderson, P., and McGuire, 1‘. C., Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats, Infect. Immun. 41, 657-665, 1983. 25. Johnson, G. C., Adams, D. S., and MC Guire, T. C.. Pronounced production of polyclonal immunoglobulin Cl in the synovial fluid of goats with caprine arthritis-encephalitis virus infection. Infect. Immunol. 41, 805-815, 1983. 26. Geballe, A. P., Ventura, P., Stowring, L., and Haase, A. ‘T., Quantitative analysis of visna vu-us replication in viva. Viro/ogy 141, 148-154, 1985. 27. Matsushima, K.. Morishita, K., Yoshimura. T., Law. S., Kobayashi, Y, s Lew, W.. Appella, L:., Kung, H. F.. Leonard, E. J., and Oppenheim. J. J., Molecular cloning of a human monocytederived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167, 1883-1893, 1988. 28. Yamauchi, K., Martinet, Y., and Crystal, R. G., Modulation of fibronectin gene expression tn human mononuclear phagocytes. J. C/in. Invest. 80, 1720-1727, 1987, 29. Momex, J. F., Martinet, Y.. Yamauchi, K.. Bitterman, P. B.. Grotendorst, G. R., Chytil-Weir. A., Martin, G. R., and Crystal, R. G.. Spontaneous expression of the c-sis gene and release of a platelet-derived growth factor-like molecule by human alveolar macrophages. J. C/in. Invest. 78, 61-66. 1986.
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30. Kennedy, P. G. E., Narayan, O., Ghotbi, Z., Hopkins, J., Gendelman, H. E., and Clements J. E., Persistant expression of Ia antigen and viral genome in visna-maedi virus induced inflammatory cells: Possible role of lentivirus-induced interferon. J. Exp. Med. 162, 1970-1982, 1985. 31. Adams, D. O., and Hamilton, T. A., Molecular transductional mechanisms by which IFNy and other signals regulate macrophage development. Immunol. Rev. 97, 5-29, 1987. 32. Schreiber. R. D., and Celada, A., The role of IFNy in macrophage activation. Lymphokines 11, 87-92, 1983. 33. Cofano, F., Comoglio. F., Landolfo, S., and Tarone, G., Mouse immune interferon enhances ifbronectin production of elicited macrophages. J. Zmmunol. 133, 3102-3106, 1984. 34. Narayan, O., Sheffer, D., Clement& J. E., and Tennekoon G., Restricted replication of lentivirus: Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages. J. Exp. Med. 162, 1954-1969, 1985. 35. Lairmore, M. D., Salvatore Butera, T., Callahan, G. N., and DeMartini, J. C., Spontaneous interferon production by pulmonary leukocytes is associated with lentivirus-induced lymphoid interstitial pneumonia. J. Zmmunol. 140, 77!&784, 1988. 36. Kennedy-Stroskopf, S., Zink, C., and Narayan, O., Pathogenesis of ovine lentivirus-induced arthritis: Phenotypic evaluation of T lymphocytes in synovial fluid, synovium, and peripheral circulation. C/in. Immunol. Immunoparhol. 52, 323-330, 1989. 37. Cordier, G., Cadore, J. L., Cozon, G., and Momex, J. F., In vivo activation of alveolar macrophages and lymphocytes in spontaneous interstitial lung disease due to the visna-maedi virus in sheep. In “Vth International Conference on AIDS.” p. 603. Montreal, 1989. [Abstract] Received September 11, 1989; accepted with revision January 9. 1990.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
In Vivo Activation
55,
355-367 (1990)
of Alveolar Macrophages Lentivirus Infection
in Ovine
GENEVI~E CORDIER,* GRBGOIRECozoN,*-t TIMOTHY GRmNLmD,*~t FRANCOIS ROCHER,t FRANCOISGUIGUEN,~ SYLVIANE GUERRET,~ JEANBRUNE,~ANDJEAN-FRANCOISMORNEX~ *INSERM V 80, CNRS VRA 1177. Vniversitk Claude Bernard (Centre de Cytojluoromktrie), H6pital Edouard Herriot, Lyon, France; TLaboratoire d’lmmunologie et de Biologic Pulmonaire, Service de Pneumologie, Hbpital Louis Pradel et Facultk de MPdecine Grange Blanche, Lyon, France; SLaboratoire Associd de Pathologie des Petits Ruminants, INRA, Ecole Nationale VSrinaire, Marcy I’Etoile, France; and flnstitut Pasteur, CNRS VRA 167, Lyon, France
Sheep infected by visna-maedi virus, a lentivirus related to the human immunodeficiency virus, develop a chronic interstitial lung disease. Since monocyte/macrophages are known to be specifically infected by visna-maedi virus, we investigated the role of macrophages in the appearance of pulmonary lesions in animals with naturally occurring disease. Alveolitis in maedi leads to a doubling in bronchoalveolar lavage total cell counts and of macrophages as compared to normal sheep. A significant increase in the relative percentage of neutrophils was also observed, accompanied by an increased spontaneous release of neutrophil chemotactic activity by alveolar macrophages of diseased animals, suggesting that they may be activated. Macrophage activation is also demonstrated by the observation of a significant (X 3) increase of spontaneous fibronectin release by alveolar macrophages from maedi lungs, and furthermore by the high level expression of major histocompatibility complex class II antigens on most of these celfs. Thus viral infection, although restricted to a small population of macrophages, is able to modulate extensive activation of macrophages in the lung. Activated macrophages release mediators likely to play a role in the development of the alveolitis and the parenchymal desorganization. These findings may be relevant to our understanding of the mechanisms by which human immunodeficiency virus infection leads to pulmonary disease other than that caused by opportunistic infections. 0 1990 Academic Press, Inc.
INTRODUCTION
Visna-maedi virus, isolated from sheep, is a member of the lentiviruses, a subfamily of nononcogenic retroviruses (1, 2). Lentiviruses are known to infect various species: goats (caprine arthritis encephalitis virus, CAEV); horses (equine infectious anemia virus); cattle (bovine immunodeticiency virus); cats (feline immunodeficiency virus, FIV); and primates (human immunodeficiency viruses, HIV-l and HIV-2 in humans and simian immunodeficiency viruses, SIV, in monkeys), While some of these viruses, e.g., HIV-I, SIV, and FIV, are associated with an immunodeficiency syndrome, all of them induce chronic and/or degenerative diseases affecting the lungs, joints, mammary glands, and the central nervous system (1, 2). In sheep, spontaneously or experimentally infected by visna-maedi virus, the primary target organ is the lung where an interstitial lung disease known as ovine progressive pneumonia or maedi develops (3-5). Lung pathology includes chronic interstitial inflammation with dense cellular infiltration, hyperplasia of smooth 355 0090- 1229FM $1.50 Copyright All rights
Q 1990 by Academic Press, Inc. of reproduction in any form reserved.
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muscle cells in the alveolar septa, slight fibrosis, peribronchial and perivascuiar lymphoid hyperplasia, and epithelial proliferation in small bronchi and bronchioles (3, 4) and (R. Loire, unpublished observations). An interstitial lung disease with similar pathological features has also been reported in infections by CAEV (6) and HIV-l (7, 8). Most lentiviruses infect cells of the monocyteimacrophage lineage in iai~j (reviewed in Refs. (1, 2)). The effect of this infection on mononuclear phagocyte functions remains controversial; decreased phagocytic (9). chemotactic (lo), and chemiluminescent (11) responses of blood monocytes have been reported in lwmans in the acquired immunodeficiency syndrome, whereas increased secretion of interleukin-1 (IL-l) by monocytes could be detected as a consequence of infection by HIV-l (12, 13) or in the presence of its products (14). Since macrophages can exert multiple functions and can modulate inflammatory processes (reviewed in Ref. (15)). some of the pathologic events observed in lentiviral in fections may be due to these cells. The interstitial lung disease due to visna-maedi virus allows the assessment of the in viva macrophage functions in the course of natural infection in the absence of opportunistic infections. By analyzing cells collected by bronchoalveolar lavage from lungs of maedi animals, we could demonstrate macrophage activation and the release of mediators involved in the pathogenesis of the interstitial lung disease. MATERIALS
AND
METHODS
Sheep Lungs
Adult sheep lungs were obtained from slaughterhouses. Lungs with macroscopic or histological evidence of parasitic infections were excluded, as were those showing hemorragic and bacterial contamination after adequate examination of bronchoalveolar lavage fluid. The diagnosis of maedi (n = 31) was based upon histological examination. Diagnostic criteria included peribronchovascular lymphoid nodules, alveolitis, and increased number of smooth muscle cells and fibroblasts (3). Control lungs (rz = 26) were defined by the absence of macroscopic and microscopic abnormal features. Bronchoalveolar Lavuge
In order to collect alveolar cells, a flexible canula was passed through the trachea and wedged into one of the main stem bronchi. Three hundred milliliters of Hank’s balanced salt solution (HBSS; BioMerieux, Charbonnieres les Bains, France) was infused and aspirated after gentle massage of the lung. The recovered liquid was immediately filtered through sterile gauze. Total cell counts were established from numeration with a hemocytometer. Differential counts of bronchoalveolar lavage cells were performed on cytocentrifuge (Cytospin, Shandon S.A., Enguy, France) smears stained using ’ ‘Diff-Quick” (Merz & Dade AG, Dtidingen, Switzerland). Cell Suspensions
Cells were separated from bronchoalveolar
lavage fluid by centrifugation
for 15
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min at 400g. Bronchoalveolar cells were washed twice in HBSS and resuspended at lo7 cells/ml in culture medium (RPM1 1640 medium supplemented with a mixture of penicillin, 100 U/ml; streptomycin, 50 t&ml; and vancomycin, 20 pg/ml). Cell viability was measured at this step by trypan blue exclusion and was consistantly over 85%. Evaluation of Neutrophil Chemotactic Activity (NCA) Generated by Alveolar Cells For evaluating the generation of NCA by alveolar macrophages, lo6 cells/ml RPM1 1640 medium without antibiotics were incubated in sterile tissue culture flasks for 3 hr in 5% CO2 in air, at 100% humidity. Supernatants were harvested by aspiration and freed of cells or debris by centrifugation at 28,000g for 10 min; they were stored at -20°C before testing. Neutrophil chemotactic activity in these supernatants was demonstrated by measuring the migration of human neutrophils in a 48well microchemotaxis chamber (Neuroprobe, Cabin John, MD) through a 3-pm micropore filter (PVPfree filter, Nucleopore, Pleasanton, CA). Human neutrophils were prepared from peripheral blood of healthy volunteers by gradient centrifugation on Mono-poly resolving medium (Flow Laboratories, Les Ulis, France) according to the supplier’s methodology. The resulting neutrophil suspensions contained more than 98% neutrophils. Aliquots (25 ~1) of supernatants to be assayed were placed in the lower wells, the filter sheet was fixed in place to separate bottom from top wells, and cells were added to the upper wells (3 x 106/ml culture medium, 40 t.~l). The chamber was incubated for 45 min at 37°C. During incubation, migrating cells penetrated the filter and remained attached to its lower surface. At the end of the incubation the filter was removed and cells which had not migrated into the pores were removed by drawing it over a wiper blade. The filter was then fixed and stained with Diff-Quick stain. Chemotactic activity is usually expressed as the number of cells counted per high power field of the stained filter. In preliminary assays we observed that the staining could be evaluated optically. Preliminary experiments showed that the light absorbance of the fixed stained cells, integrated with a spectrophotometer (Vernon PHI 6, Paris, France) over each spot corresponding to a well, increased regularly with the number of cells present over the range of 0 to 150 x lo3 under experimental conditions (data not shown). We thus used light absorbance as an index of migration. The results were standardized between assays by defining 100 migration units as the maximal light absorbance obtained using formylL-methionyl-L-leucyl-L-phenylalanine oligopeptide (Calbiochem, FranceBiochem, Meudon, France) at lop7 M as an attractant (optimal concentration under our experimental conditions). The relative attraction of culture supernatants was expressed as units per milliliter corresponding to the activity released by lo6 macrophages per milliliter. Fibronectin Release by Alveolar Macrophages The release of fibronectin by alveolar cells was measured in culture supematants. Based on the kinetics of production observed by Rennard et al. (16) for
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human alveolar macrophages, 5 x lo6 cells in 5 ml culture medium were incubated for 21 hr at 37°C in 5% CO2 in air at 100% humidity. In some experiments, alveolar cells from healthy lungs were cultured in the presence of lipopolysaccharide (LPS; Difco laboratories, Detroit, MI) at 10 l&ml or freshly opsonized zymosan particles (Sigma, St. Louis, MO) at 5 x lo6 particles/ml. Cell-free supernatants obtained by centrifugation (28,OOOg, 10 min) were stored frozen at - 20°C until used. The fibronectin released in supernatants was evaluated using a sandwich ELISA relying on the affinity of fibronectin for gelatin on the one hand and the binding of anti-tibronectin antibodies on the other. Purified rabbit anti-human fibronectin antibodies were prepared at the Institut Pasteur de Lyon (17). Crossreactivity with purified sheep fibronectin was demonstrated by Ouchterlony immunoprecipitation; in addition, no reactivity was demonstrated toward fibronectin-depleted sheep serum after a two-step affinity-chromatography purification (data not shown). The microplate (Nunc-Immuno, Roskilde, Denmark) was coated with a gelatin solution prepared by warming 2.5 mg/ml of gelatin (Bio-Rad Laboratories, Richmond, CA) in carbonate-bicarbonate buffer (Na,CO,, 15 n&f; NaHCO,, 35 mM; pH 9.3) for 10 min at 90°C. Two hundred microliters of this solution was delivered per well and incubated for 18 hr at room temperature. After five washings with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma) (PBS-Tween), each well received 200 ~1 of alveolar cell supernatant for a further incubation of 2 hr at room temperature. Then, the supernatant was discarded and the wells were again washed five times with PBS-Tween. To each well was then added 200 l.~l of a 1:250 dilution of the anti-fibronectin antibodies. After incubation for 1 hr and washing (~5) with PBS-Tween, 200 ~1 of peroxidaseconjugated goat anti-rabbit Ig antibodies (Biosys, Compibgne, France) at optimal dilution (1:4000) in the presence of 0.1% goat serum (Seralab, Sussex, England) was added for 1 hr. The excess reagent was discarded and the plates were washed five times with PBS-Tween. Finally the plates were incubated for 15 min with 200 p.l/well of substrate O-phenylenediamine dihydrochloride (Sigma). The enzymatic reaction was stopped by adding 100 pi/well of H,SO, (2 N) and the absorbance at 492-650 nm was read in an ELISA processor (SLT210, Kontron AG, Zurich, Switzerland). The standard curve for dosage in each test was obtained using serial dilutions (1:2000 to l:lOO,OOO) of a pooled normal sheep sera preparation stored frozen at - 20°C until use. This preparation contains 298 &ml of fibronectin as assessed by Dr. S. Bozena-Begin (University of Sherbrooke, Sherbrooke, Canada). Amounts of fibronectin in the test supernatants were evaluated from the mean value of triplicate measures. Results are expressed as nanograms per milliliter of tibronectin released by lo6 macrophages per 24 hr. Macrophage
Membrane
Antigen
Expression
Indirect immunofluorescence was used to determine membrane antigenic expression by alveolar cells. Sheep major histocompatibility complex (MHC) class II antigens were identified using two specific monoclonal antibodies: SBU-II (clone 28-l), a mouse IgGl anti monomorphic MHC class II antigen (18) supplied
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by Dr. Brandon (University of Melbourne, Australia), and SW (clone 73.2), a rat Ig anti-monomorphic MHC class II antigen (19) kindly donated by Dr. Myasaka (Base1 Institute for Immunology, Switzerland). In addition, hybridoma 175, a mouse IgM K, anti-sheep myeloid and erythroid cells (19), was used to identify alveolar cells. A purified preparation of FITC-conjugated goat IgG anti-mouse IgG + IgM (FITC-GAMIG; Biosoft, Paris, France) was used as the second step reagent. Samples of lo6 cells were incubated for 30 min in 50 ~1 primary antibody preparation at the optimal dilution. They were then washed twice with PBS containing 15 mM NaN, plus 1% BSA (PBS-BSA-Azide) and the pellets were resuspended in 50 ~1 of the appropriate dilution of FITC-GAMIG for a further 30 min. Cells were washed twice and fixed in 700 ~1 of PBS-BSA-Azide with 1% formaldehyde. Control samples incubated with FITC-GAMIG alone were included in each series. Cells were analyzed in a Cytofluorograf 50 H (Ortho Instruments, Westwood, MA) equipped with a 5 W Argon-ion laser and interfaced with an interactive computer developed in this laboratory (20). The 488-pm laser line was used at 400 mW for excitation. Three parameters were recorded for each cell: the forward angle light scattering (FAS) which is related to cell diameter, the right angle light scattering (RAS) which gives information on the internal structural properties of the cell, and green fluorescence related to the fixation of antibodies. Preliminary experiments showed that using a log scale on both FAS and RAS parameters it was possible to discriminate between various clusters. Analysis of the correlated fluorescence for each cluster in sample aliquot stained with 175 allowed the identification of macrophages (175-positive, high FAS and RAS). In contrast, lymphocytes were shown to be 175-negative cells with low FAS and RAS whereas neutrophils were characterized as 175-positive cells with intermediate FAS and RAS. Fluorescence histograms were recorded using the gating on FAS and RAS parameters. For each sample, 10,000 to 20,000 cells were accumulated in order to get a fluorescence histogram with at least 5000 cells within each gate. The percentage of positive cells was calculated for biphasic distribution by counting cells above a threshold established on control cells. When the test and control histograms overlapped, the threshold was set at the channel above the overlapping area. Fluorescence intensity was expressed as arbitrary units. Evidence of Viral Infection The presence of lentivirus in alveolar macrophage specimens was demonstrated by the occurrence of a cytopathic effect following cocultivation with permissive cells. Briefly, bronchoalveolar lavage cells were resuspended in culture medium at 2-3 x 106/ml, then l-2 x 106/ml of freshly trypsinized ovine skin fibroblasts (ID05 strain) were added and the coculture was incubated in 25-cm* Falcon flasks. Cultures were examined every day by phase contrast for evidence of cytopathic effect. Viral infection was demonstrated by the appearance by Days 10-30 of culture of syncytia (>g nuclei/cell) observed following fixation and staining of the fibroblasts with May-Grumwald-Giemsa. Negative cultures were monitored for over 60 days.
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Statistical Analysis Results are presented as means t SEM. Statistical Student’s two-tailed test.
analysis was made using
RESULTS
Accumulation
of Inflammatory
Cells within the Alveolar
Spaces
Maedi is characterized by an alveolitis. This was demonstrated by direct sampling of alveolar cells using bronchoalveolar lavage. Both total and differential cell counts were evaluated (Table I), the number of alveolar cells is significantly increased in sheep with maedi (n = 12) when compared to healthy controls (n = 15). In lavages from normal lungs (n = 19). alveolar cells were mainly macrophages, the distribution was modified in lavages from maedi lungs (n = 20) with a significant increase in the percentage of neutrophils (Table 1). Although the percentage of macrophages was decreased (Table l), the absolute macrophage counts were significantly increased from 0.9 x 106/ml 2 0.1 in healthy lungs to 1.8 x 106/ml + 0.5 (P < 0.05) in the maedi ones. There was no significant change of the lymphocyte percentage (Table 1). Generation
of NCA by Alveolar
Macrophages
To investigate a possible cause for the increased neutrophil content within the alveolar spaces, we evaluated the generation of NCA by alveolar macrophages. The chemotactic activity of macrophages culture supernatants was assessed using human neutrophils as indicator cells. To ensure that our culture conditions allowed the generation of NCA and that human neutrophils could react to it, we tested supernatants from normal sheep alveolar cells stimulated with zymosan particles and LPS. These supernatants repeatedly induced migration of human neutrophils as shown by the migration values observed: 49.5 -C 2.0, 38.2 + 6.5, and 14.3 k 3.8 units/ml, respectively, for zymosan. LPS, and control medium in seven separate experiments (Fig. 1). Supernatants of bronchoalveolar lavage cells from maedi lungs (n = 18) cultured in the absence of stimulants were compared to those obtained from healthy lungs (n = 15). The results (Fig. 1) indicate that alveolar cells from infected animals spontaneously produce increased amounts of TABLE CELLULARCOMPOSITION
Normal Maedi
I
OF BRONCHOALVEOLAR
Cell counts” (lo6/ml)
Macrophage@
1.7 k 0.3 (0.6-5.4) 3.7 L 1.1 (0X-12.0)
78.0 + 2.0 62.0 r 4.0
5.0 t 1.0 13.0 t 3.0
P < 0.05
P < 0.05
P < 0.05
u Means 2 SEM (range). b Expressed as percentage of total cells. ’ Expressed as percentage of macrophages. ’ Not significant.
Neutrophils”
LAVAGE
Lymphocytes” 15.0 t 2.0 18.0 -t 3.0 nsJ
MHC clas:, II positive macrophages”,’ IS.5 _t 4.0 (U-37) SO.1 + 4.5 (24-86)
P i 0.05
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60 1 50-
40-
30-
20-
lo-
O-
normal
maedi
IlOriIXil
+LPS
normal +Zymosan
FIG. 1. Generation of NCA by alveolar macrophages from healthy (n = 19) and maedi (n = 18) lungs. Supematants were prepared from lo6 cells/ml incubated for 3 hr in culture medium. Migration indexes were measured as described under Materials and Methods. Bars indicate the mean value (2 1 SEM) of migration index observed for each group.
NCA (28.1 ? 4.7 units/ml vs 16.6 + 3.1 units/ml, P < 0.05) giving one possible explanation for the increased proportion of neutrophils in the alveolar spaces. Fibronectin Release by Alveolar Macrophages
Fibronectin is a mediator which could modulate some of the lung parenchymal lesions; we thus evaluated the spontaneous release of fibronectin by alveolar macrophages. Fibronectin was measured in culture supernatants of alveolar cells from maedi lungs (n = 11) or from normal lungs (n = 14), with or without LPS or zymosan stimulation (Fig. 2). It can be seen that the maedi bronchoalveolar lavage macrophages spontaneously released three times as much libronectin as did the normal cells (11.4 + 3.2 r&ml vs 3.5 ? 0.9 rig/ml per 24 hr, P < 0.05). Interestingly enough, unlike the situation for NCA, stimulation of normal macrophages did not increase their production of libronectin (Fig. 2). This suggests that different mechanisms are involved in the control of the production of these two factors. MHC Class II Antigen Expression by Alveolar Macrophages
Although MHC Class II antigens are constitutively expressed by macrophages, their level of expression is tightly regulated. In suspensions prepared from normal lungs (n = 15) the percentage of MHC class II antigen-positive macrophages was low (15.5 + 4.0, Table 1) using either the SW mAb or the SBU-II mAb. both specific for a monomorphic epitope of sheep MHC class II antigens. In suspensions prepared from maedi lungs (n = 13), we observed an increase in the percentage of labeled cells (50.1 2 4.5%; P < 0.05 when compared to healthy specimens). A change in antigen density could also be observed as depicted by the shift of the fluorescence profile (Fig. 3).
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ET AL.
maedi
normal +I,PS
normal +zymosan
FIG. 2. Fibronectin release by alveolar macrophages. Fibronectin was assayed by ELlSA as described under Materials and Methods. Supernatants were prepared by culture of 5 x IO’ cells in 5 ml culture medium for 21 hr from maedi (n = 12) and healthy lungs without (n = 15) and with stimulation by LPS (50 &ml, n = 13) and zymosan particles (5 x lO?ml. n = 8). Bars indicate the mean value (21 SEM) of tibronectin (r&ml) released per IO’ cells/24 hr for each group.
Evidence of Lentiviral Infection Alveolar macrophages were cultured with ovine tibroblasts in order to detect lentiviral infection. No cytopathic effect was observed when fibroblasts were cultured alone. Similarly, macrophages from 19 microscopically normal lungs were cultured with fibroblasts without evidence of syncitia for up to 2 months (three to four passages). In contrast, macrophages from 11 out of 12 maedi lungs led to syncitia formation. Further characterization of these isolates included the demonstration of a reverse transcriptase activity (two cases, data not shown) and the presence of viral particles by electron microscopy examination (four cases, data not shown). Thus alveolar macrophages collected from maedi lungs harbored a lentivirus. DISCUSSION
Lentiviruses induce chronic inflammatory and/or degenerative disorders within multiple organs. In sheep infected by visna-maedi virus the lung is the main target organ. Most of the attention has so far focused on the slow development of the disease and its delay in appearance. Although the virus is known to infect the cells of the monocyte/macrophage lineage specifically, no s:tndies have investigated the effect of lentiviral infection on macrophage functions. In this study, using bronchoalveolar lavage to gain access to the inflammatory cells at the site of the disease, we can characterize the alveolitis process occurring in spontaneously infected animals. We also evaluate the ability of alveolar macrophages to produce mediators involved in the attraction and the proliferation of the various cell types observed in the affected lung. Distribution of alveolar cells in healthy lungs is similar to that previously described (21). In maedi lungs, diagnosed on pathological grounds as described
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Maedi
fluorescence intensity FIG. 3. Fluorescence profiles of alveolar macrophages. Cells prepared from healthy (left) and maedi (right) lungs were stained for MHC class II antigens using the SBU-II mAb (A) and for a myeloid antigen using the 175 mAb (B). Histograms were obtained from at least 5 x lo3 macrophages gated according to their RAWFAS characteristics. The change in antigen density is demonstrated by the shift of the histograms.
elsewhere (3) besides the pathological evidence of alveolar wall inflammation there is a luminal alveolitis as shown by the significant increase in the number of alveolar cells. Cells comprising this alveolitis are mainly macrophages and neutrophils without significant increase in the percentage of lymphocytes. The increased macrophage content in the alveoli and the dense infiltration by neutrophils are known from histological studies in animals with spontaneous advanced maedi (3). As has previously been shown in the human (22), cells collected by bronchoalveolar lavage reflect the inflammatory process occurring in the lung and we therefore used bronchoalveolar lavage cells for the assessment of the macrophage function in the lung. Evidence of Macrophage Activation
In agreement with the observed neutrophilic alveolitis we find an increased spontaneous NCA release by alveolar macrophages from maedi lungs. It is observed in the absence of exogenous stimulatory agents. A variety of agents, including microorganisms, noninfectious particles, and immune complexes, are able to induce the generation of NCA (23). A role for immune complexes remains a distinct possibility since viral antigen production continues in antibody positive
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sheep, and production of IgGl in synovia of CAEV-infected goats probably represents a local response (24, 25). No antibodies to visna-maedi virus were found in one study of bronchoalveolar lavage liquid of maedi sheep (26), suggesting a low probability of local complex formation; however, our recent observations suggest that anti-viral antibodies may sometimes be found in bronchoalveolar lavage fluids (D. Ecochard, unpublished data). One neutrophil chemotactic factor has been shown to be delicately regulated. This recently described monocytederived neutrophil chemotactic factor is induced by II- 1 and tumor necrosis factor but not by interferon-y (IFN-y) (27). This would suggest complex interactions between infected and noninfected cells at the site of the disease. Fibronectin is able to stimulate the growth of fibroblasts thus playing a role in modulating damage and repair in inflammatory processes (16, 22, 28). We demonstrate an increased spontaneous release of fibronectin by alveolar macrophages from maedi lungs. Fibronectin is released in small amounts by alveolar macrophages from normal sheep lungs, LPS and immune complexes were unable to increase fibronectin secretion as previously shown in humans (28). Fibronectin production is regulated by steady-state levels of mRNA; currently no information is available concerning the controlling elements for fibronectin gene expression or the mechanism modulating the fibronectin mRNA life span. In maedi lungs most of the alveolar macrophages express high levels of MHC class II antigens. A small percentage of alveolar macrophages from normal iungs express these antigens in agreement with prevous observations (7). Modulation of class II antigen expression has been largely documented among changes occurring in activated macrophages (15). Increased expression of MHC class II antigens by macrophages of maedi animals together with the above observations, i.e., increased release of NCA and fibronectin, support the existence of an activation process within the lung in maedi animals. The mediators released in situ by activated alveolar macrophages are likely to play a role in the development of the lung pathologic changes as shown in human interstitial lung diseases (16, 22, 23, 29). Mechanisms
of Macrophage
Activation
The mechanisms leading from macrophage infection to macrophage activation remain to be elucidated. In this study, alveolar macrophages from animals with histologically proven maedi harbor a visna-maedi-like virus and normals do not. A direct activation of macrophages by visna-maedi virus could therefore be possible. Two lines of evidence argue against this: (i) whereas in our study up to 50% of macrophages are activated as assessed by their increased expression of MHC class II antigens, only 0.1 to 1% macrophages are known to express virus in infected animals (reviewed in Ref. (1)) and (ii) in vitro infection of macrophages does not lead to an increased expression of MHC class II antigens (30). An indirect mechanism of activation is more probable. Macrophage activation is under the control of signals operationally defined as macrophage-activating factors. Most of them are released by activated lymphocytes. One of the most potent in vivo and in vitro is IFN-y (31) which is well known as an inducer of Ia antigen expression (31, 32) and is able to enhance the secretion of various proteins including, in the mouse, the production of tibronectin (33). Such a cellular interac-
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tion is likely to occur since (i) an interferon is spontaneously released by lymphocytes cocultured with in vitro-infected macrophages (34) and by lymphocytes collected from lymph nodes of experimentally infected lambs (39, moreover it is present in synovial fluid from naturally infected sheep (36) and (ii) we have demonstrated activated alveolar T lymphocytes in maedi lungs (37). In conclusion, the interstitial lung disease observed in spontaneous visna-maedi virus infection is mediated by macrophage activation. Activated macrophages show increased production of mediators able to modulate the interstitial lung disease in situ. Macrophages are likely to be activated through complex cellular interactions as a consequence of viral infection. Since the genomic organization and the basic biological features of the host-virus interactions are remarkably similar for visna-maedi virus and HIV-I, the interstitial lung disease due to visnamaedi virus offers a useful model for the study of lentiviral-induced inflammatory disease in humans. ACKNOWLEDGMENTS The authors thank Mr. Roussin and Mr. Lefevre for providing them with animal lungs; Dr. M. Fontaine for virus isolation; Dr. R. Loire for performing the pathological examinations; Dr. S. BozenaBegin and Dr. A. Cantin for tibronectin assay in the reference preparation; Dr. G. Caillot for the preparation of purified sheep tibronectin and Bbronectin-depleted sheep serum; Dr. M. Myasaka for the generous gift of SW and 175 mAb; and Mss. V. Pelamatti and C. Quintin for expert secretarial assistance. They are especially grateful to Professor J. P. Revillard for advice and valuable discussion during this work. This work was supported in part by grants from: INSERM (CRE 865011); Minis&e de la Recherche et de I’Enseignement Superieur (Programme National de Recherche sur le SIDA); Minis&e de la Recherche et de la Technologie (FRT); Universite Claude Bernard, Departement de Biologie Humaine et Fact&C Grange Blanche; Fond Special des Comites Departementaux de Lutte contre les Maladies Respiratoires (87/MR21, 88 MR/13); Hospices civils de Lyon; Institut Mtrieux, Marcy I’Etoile (France).
REFERENCES 1. Haase. A. T., Pathogenesis of lentivirus infections. Nature (London) 322, 130-136, 1986. 2. Narayan, 0.. and Clements, J. E., Biology and pathogenesis of lentiviruses. J. Gen. Viral. 70, 1617-1639. 1989. 3. Georgsson, G., and Palsson, P. A., The histopathology of Maedi: A slow viral pneumonia of sheep. Vet. Pathol. 8, 63-89, 1971. 4. Lairmore, M. D., Rosadio, R. H., and De Martini, J. C., Ovine lentivirus lymphoid interstitial pneumonia: Rapid induction in neonatal lambs. Amer. J. Pathol. 125, 173-181, 1986. 5. Momex, J. F., Greenland, T., Cordier, G., Rocher, F., Cozon, G., Loire, R., Patet, J., Guiguen, F., Cadore, J. L., and Brune, J., Diffuse interstitial lung disease in sheep caused by Maedi/Visna virus. In “The Alveolar Macrophage” (C. Sorg, Ed.), pp. 96100. Regensbers and Bierman, Munster. 1988. 6. Straub. 0. C., Caprine arthritis encephalitis: A model for AIDS? Intervirology 30, 45-50. 1989. 7. Marchevsky, A., Rosen, M., Crystal, R., and Kleinerman, J., Pulmonary complications of the acquired immunodeficiency syndrome: A clinicopathologic study of 70 cases. Hum. Pathol. 16, 659-670, 1985. 8. Joshi, J., Oleske, J., Minnefor, B., Saad. S., Klein. K., Singh, R., Zabala. M., Dadzie, C., Simpser, M., and Rapkin, R., Pathologic pulmonary findings in children with the acquired immunodeficiency syndrome: A study of ten cases. Hum. Pathol. 16, 241-246, 1985. 9. Estevez, M. E., Ballard, I. G., Diez, R. A., Planes, N., Scaglione, C., and Sin. L., Early defect of phagocytic cell function in subjects at risk for acquired immunodeficiency syndrome. &and. J. Immunol. 24, 215-221, 1986.
366
CORDIER
ET
AL.
10. Smith, P. D., Chura, K., Masur, H., Lane, H. C., Fauci, A. S., and Wahl, S. M.. Monocyte function in the acquired immunodeficiency syndrome defective chemotaxis. J. C/in. invest. 74, 2121-2124, 1984. Il. Braun. D. P., Kessler, H., Falk, L., Paul, D.. Harris, J. E., Blaauw, B., and Landay A., Monocyte functional studies in asymptomatic, human immunodeficiency disease virus (HIV)-infected individuals. 1. C/in. Immunol. 8, 486-494, 1988. 12. Roux-Lombard, P., Modoux, C., Cruchaud, A., and Dayer, J. M.. Purified blood monocytes from HIV-l-infected patients produce high levels of TNF u and IL-I. C/in. Immunol. Immunopnthol. 50, 374-384, 1989. 13. Folks, T. M.. Justement, J., Kinter, A., Dinarello, C. A., and Fauci A. S.. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238, 800-802, 1987. 14. Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Hare]-Bellan, A.. and Farrar. W. L,.. Human immunodeticiency virus glycoprotein (gp 120) induction of monocyte archidonic acid metabolites and interleukin 1. hoc. Nat/. Acad. Sci. USA 86, 621-625. 1989. 15. Adams, D. O., and Hamilton, T. A., The cell biology of macrophage activation. Anna, Ret. lmmunol.
2, 283-313,
1984.
16. Rennard, S. I., Hunninghake, G. W., Bitterman, P. B.. and Crystal, R. G., Production ot fibronectin by the human alveolar macrophage: Mechanism for the recruitment of fibroblasts to sites of tissues injury in interstitial lung diseases. Proc. Nat/. Acad. Sci. USA 78,7147-7151, 1981. 17. Grimaud, J. A.. Druguet M.. Peyrol, S., and Guerret. S., Collagen. In “Methods of Enzymatic Analysis, Proteins and Peptides” (H. U. Bergmeyer, Ed.), Vol. 9. pp. 186195, VCH Publishers, Weinheim, 1986. 18. Puri, N. K., Mackay, C. R., and Brandon, M. R., Sheep lymphocyte antigens (OLA). Il. MaJor histocompatibility complex class II molecules. Immuno/ogy 56, 725-734, 1985. 19. Miyasaka, M., McClure, S., Hein, W. R., and Tmka, Z., Differentiation antigens in lymphohemopoietic system of the sheep. In “Differentiation Antigens in Lymphohemopoietic Tissues” (M. Miyasaka and 2. Tmka, Eds.), pp. 499-523, Dekker, New York/Base]. 1988. 20. Momex, J. F., Cordier, G.. Pages, J., Lefebvre. R., Revillard, J. P.. Vergnon. J. M.. Vincent. M., Cordier, J. F., and Brune, J., Pulmonary sarcoidosis: Flow cytometry measurement of lung T cell activation. J. Lab. Clin. Med. 105, 70-76, 1985. 21. Rola-Pleszcynski. M., Sirois, P., and Begin, R., Cellular and humoral components of bronchoalveolar lavage in the sheep. tung 159, 91-99, 1981. 22. Keogh. B. A., and Crystal. R. G., Alveolitis: The key to the interstitial lung disorders. %NU~ 37, I-10. 1982. 23. Hunninghake, G. W., Gadek, J. E., Fales, H. M., and Crystal. R. G.. Human alveolar macrophage-derived chemotactic factor for neutrophils. Stimuli and partial characterization. J. C/in. Invest. 66, 473-483, 1980. 24. Johnson, G. C.. Barbet, A. F., Klevjer-Anderson, P., and McGuire, 1‘. C., Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats, Infect. Immun. 41, 657-665, 1983. 25. Johnson, G. C., Adams, D. S., and MC Guire, T. C.. Pronounced production of polyclonal immunoglobulin Cl in the synovial fluid of goats with caprine arthritis-encephalitis virus infection. Infect. Immunol. 41, 805-815, 1983. 26. Geballe, A. P., Ventura, P., Stowring, L., and Haase, A. ‘T., Quantitative analysis of visna vu-us replication in viva. Viro/ogy 141, 148-154, 1985. 27. Matsushima, K.. Morishita, K., Yoshimura. T., Law. S., Kobayashi, Y, s Lew, W.. Appella, L:., Kung, H. F.. Leonard, E. J., and Oppenheim. J. J., Molecular cloning of a human monocytederived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167, 1883-1893, 1988. 28. Yamauchi, K., Martinet, Y., and Crystal, R. G., Modulation of fibronectin gene expression tn human mononuclear phagocytes. J. C/in. Invest. 80, 1720-1727, 1987, 29. Momex, J. F., Martinet, Y.. Yamauchi, K.. Bitterman, P. B.. Grotendorst, G. R., Chytil-Weir. A., Martin, G. R., and Crystal, R. G.. Spontaneous expression of the c-sis gene and release of a platelet-derived growth factor-like molecule by human alveolar macrophages. J. C/in. Invest. 78, 61-66. 1986.
ACTIVATED
MACROPHAGES
IN
LENTIVIRAL
INFECTION
367
30. Kennedy, P. G. E., Narayan, O., Ghotbi, Z., Hopkins, J., Gendelman, H. E., and Clements J. E., Persistant expression of Ia antigen and viral genome in visna-maedi virus induced inflammatory cells: Possible role of lentivirus-induced interferon. J. Exp. Med. 162, 1970-1982, 1985. 31. Adams, D. O., and Hamilton, T. A., Molecular transductional mechanisms by which IFNy and other signals regulate macrophage development. Immunol. Rev. 97, 5-29, 1987. 32. Schreiber. R. D., and Celada, A., The role of IFNy in macrophage activation. Lymphokines 11, 87-92, 1983. 33. Cofano, F., Comoglio. F., Landolfo, S., and Tarone, G., Mouse immune interferon enhances ifbronectin production of elicited macrophages. J. Zmmunol. 133, 3102-3106, 1984. 34. Narayan, O., Sheffer, D., Clement& J. E., and Tennekoon G., Restricted replication of lentivirus: Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages. J. Exp. Med. 162, 1954-1969, 1985. 35. Lairmore, M. D., Salvatore Butera, T., Callahan, G. N., and DeMartini, J. C., Spontaneous interferon production by pulmonary leukocytes is associated with lentivirus-induced lymphoid interstitial pneumonia. J. Zmmunol. 140, 77!&784, 1988. 36. Kennedy-Stroskopf, S., Zink, C., and Narayan, O., Pathogenesis of ovine lentivirus-induced arthritis: Phenotypic evaluation of T lymphocytes in synovial fluid, synovium, and peripheral circulation. C/in. Immunol. Immunoparhol. 52, 323-330, 1989. 37. Cordier, G., Cadore, J. L., Cozon, G., and Momex, J. F., In vivo activation of alveolar macrophages and lymphocytes in spontaneous interstitial lung disease due to the visna-maedi virus in sheep. In “Vth International Conference on AIDS.” p. 603. Montreal, 1989. [Abstract] Received September 11, 1989; accepted with revision January 9. 1990.