Increased Macrophage Division in the Synovial Fluid of Goats Infected with Caprine Arthritis-Encephalitis Virus

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 157,.6. JUNE by The University of Chicago. All rights reserved /88/ $01.00 Increased Macrophage Division in the Synovial Fluid of Goats Infected with Caprine Arthritis-Encephalitis Virus Mark A. Jutila and Keith L. Banks From the Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington Macrophages are a major component of the arthritic lesions induced by the lentivirus caprine arthritis-encephalitis virus (CAEV). Using autoradiography and the appearance of mitotic figures to detect dividing macrophages, we found that 2.10/0 ± 0.2% of synovial fluid macrophages from uninfected goats are dividing and that after infection with CAEV the percentage increases three- to sixfold. The enhanced macrophage division was not associated with increased dividing of blood monoblasts. The amount of macrophage division correlated with two measures ofarthritis: joint swelling and the number ofsynovial fluid macrophages. Induction ofan immuneresponse in the jointsof CAEV-infected goats increased the number of dividing macrophages. The synovial fluid of infected animals was mitogenic for macrophages from infected animals in amounts that correlated with the amount of macrophage division occurring in the joints. Activated lymphocytes produced nondialyzable lymphokines mitogenic for macrophages from CAEV-infected goats but not from uninfected goats. These results suggest that in situ macrophage division contributes to the lesions induced by CAEV and that infection leads to greater responsiveness of macrophages to mitogenic factors produced by lymphocytes. Lentiviruses constitute a nononcogenic subfamily of retroviruses that cause slowly progressing diseases of the CNS, lungs, and joints in sheep and goats [1-7]. The humanimmunodeficiency virus (HIV) has been shown to have genomic and antigenic similarities to the sheep and goat lentiviruses and indeed may be the first-described human lentivirus [8~10]. Caprine arthritis-encephalitis virus (CAEV), the prototype of goat lentiviruses, primarily causes neurological disease in young goats [2], and, in older goats, causes an arthritis morphologically similar to human rheumatoid arthritis [1, 4, 5, 11]. The lesions caused by CAEV infection are accumulations oflymphocytes, macrophages, and a few plasma cells that replace normal tissue and lead to arthritis or encephalitis [1-3, 5, 11, 12]. Because CAEV and other lentiviruses can infect Received for publication 17 August 1987 and in revised form 5 January This work was supported by grant AM from the National Institute ofarthritis, Diabetes, Digestive and Kidney Diseases and by USDA Agricultural Research Service Cooperative Agreement 58-9AHZ We thank J. L. Carlson and Linda rton for technical assistance and Frank Michaels for helpful discussions. Please address requests for reprints to Dr. Keith L. Banks, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington macrophages [1, 13-16] and because of the role of macrophages in the virus-induced lesions, we initiated a series ofstudies to examine tissue macrophage and monocyte functions. Altered macrophage division in synovial fluid (SF) during CAEV infection could affect macrophageaccumulationin the lesions and expression of virus, because division of macrophages affects the expression of other retroviruses [17, 18]. In this study we found that synovial macrophages from infected goats divide more than macrophages from uninfected goats and thatthe amount of division was associated with the severity of the arthritis. In addition, the macrophages of infected goats were stimulated to divide when cultured with SF from arthritic joints and supernatants from cultures of stimulated lymphocytes. These results suggest that a portion of the lesions caused by CAEV were due to increased macrophage division, which possibly occurred through increased response of virus-infected macrophages to mitogenic factors produced by lymphocytes. Materials and Methods Experimental animals and inoculationsof virus. The prototype strain ofcaev (75G63) was cloned, propagated in primary fetal goat synovial cell cultures as previously described [19], and used to in- 1193

2 1194 lutila and Banks oculate the goats used in these studies. All goats were of the Saanen breed. Groups of differently aged goats were infected and manipulated in the following manner: Four goats born in 1981 wereinoculated iv and intraarticularly (ia) at 10 or 11 d of age with 0.5 ml of CAEV (1Q61CID s<lml). Four age-matched controls were inoculated in a similar manner by using processed medium from uninfected synovial cell cultures. Eight goats born in 1982 and 1983 were fed 5 x 10 6 TCID so ofcaev/ml in milk, and eight control goats were fed processed control medium at 30 d of age. Five goats born in 1984 were infected ia with 10 7 TCID so ofcaev/ml at six months ofage. Five age-matched control goats were inoculated with processed control medium. Twenty-two goats born in 1983 and 1984, half of which were given CAEV iv, were immunized, and subsequently, antigeninduced arthritis (AlA) was triggered by using methylated human serum albumin (mhsa). The full description ofthe AlA experimental protocol is contained in our previous report [20]. Briefly, 12 twomonth-old goats born in 1983 were immunized sc in the first week with 1 mg of mhsa in 1 ml of complete Freund's adjuvant and in the second week with 1 mg of mhsa in saline. During the second week, six goats weregiven 10 6 TCID so ofcaev/ ml iv and were fed 5 x to 6 TCID so of CAEV/mL in milk. The remaining six goats were given processed control medium iv and in milk. During the sixth week, all goats received ia injections of 200 Ilg of mhsa in the left carpal joint and an ia injection of saline in the right carpal joint. The same experiments were done with 10goats born in 1984,except immunization began at five months ofage. Clinical arthritis was determined by measuring the circumference ofeach carpus and metacarpus and by calculatinga carpal/metacarpal ratio, as previously described [20]. SF analysis. SF samples were aspirated from the left and right carpal joints, diluted 1:10 in DMEM (Dulbecco's MEM; GIBCO, Grand Island, NY) plus 2070 fetal bovine serum, and centrifuged at 450 g for 30 min at 4 C to isolate the SF cells. The cells were washed in calcium-magnesium-free HBSS (CMF HBSS), counted, characterized morphologically by nonspecific esterase and Wright's-stained cytosmears, and characterized functionally by adherence on plastic Lablek'" slides (Miles Scientific, Naperville, Ill) and by phagocytosis of yeast. Collection ofperipheral blood monocytes, lymphocytes, and mammary macrophages. Peripheral blood mononuclear leukocytes (PBMLs) were collected from heparinized blood obtained from infected and uninfected goats by isopycnic centrifugation [21]. Monocytes were separated from the PBMLs by subsequent adherence on plastic LabTek slides, as done during autoradiography (described below). Mammary macrophages from infected and uninfected goats were obtained by inserting a sterile catheter into a teat that had been bathed with ethanol, and 5-10 ml of mammary fluid was aspirated into a 10-mL syringe. The fluid was diluted 1:3 in CMF-HBSS, and the cells were pelleted by centrifugation at 400 g. Cell types were determined from cytospin preparations by using Wright's stain, and the resident mammary gland cell population normally consisted of macrophages, as previously reported [22]. In other experiments, PBMLs from uninfected goats were adjusted to 10 6 cells/ml in RPMI 1640 plus fetal bovine serum and culturedfor four days in the presence or absence of 2 ug of concanavalin A (Con A) under CO 2 and at 37 C. Previous studies have shown these conditions to be optimal for proliferation of goat lymphocytes in vitro [23]. After culture the supernatant fluids were collected, and portions were dialyzed for three days against a to-fold excessof RPMI 1640and then frozen at - 20 C until tested for macrophage mitogenic activity. Assays of macrophage division. (1) Mitotic index. The mitotic index (MI) was obtained as follows: Cytospin preparations of cells obtained from the joint or peripheral blood and incubated with or without yeast were stained by Wright's stain or stained for esterase content and examined for mitotic figures. The percentage of mitotic figures in 5000 macrophages was determined and recorded as the MI. (2) Autoradiography. Autoradiography was done to determine the percentage of macrophages synthesizing DNA. DNA labeling occurred either in vitro or in vivo. The in vitro-labeling assays were done by culturing macrophages in 0.4 ml of Iscove's modified DMEM (IS [24]; Sigma, St. Louis) and adding 0.25 IlCi of [3H]thymidine (6.7 Ci/mmol; New England Nuclear, Boston, Mass) and lq7 yeast cellsto the macrophages. These procedures were performed in wells on plastic slides (LabTek; Miles Laboratories) for 4 h. T!J.e slides were then vigorously washed in PBS and allowed to dry. After removing the plastic side walls, the slides were dipped

3 Increased Macrophage Division in CAE 1195 in Kodak NTB 2 liquid emulsion (Eastman Kodak, Rochester, NY), exposed for three days at 4 C, developed in Kodak 0-19 developer for 3 min, fixed (Kodak fixer), and stained by Wright's stain or stained for esterase content. The percentage ofdividing macrophages, indicated by detecting ~12 silver grains over their nuclei, was determined by counting a total of 500 cells that were adherent, had phagocytized greater than four yeast cells, were positive for nonspecific esterase, and had morphological characteristics of macrophages. To detect in vivo-labeled, DNA-synthesizing macrophages, we injected 12 uci of [3H]thymidine into the joints of four four-year-old goats infected with CAEV at 10 d ofage and two four-year-old uninfected goats, and 0.5 ml of SF was aspirated 0.5 h later. SF macrophages were harvested as described above and in-. cubated with yeast in plastic LabTek slides for 3-4 h to allow for adherence and phagocytosis; the autoradiography was then performed as described above. (3) The 96-well macrophage division assay. The 96-well macrophage division assay was performed as follows: Mammary macrophages were incubated for 2-3 h on plastic petri dishes (60 x 15 mm) at 37 C under CO 2 ; the nonadherent cells were washed away; and the adherent macrophages were removed from the plastic by cold shock, incubation in 0.01% EDTA, and gentle scraping with a rubber policeman. The percentage of macrophages was determined by phagocytosis of yeast and by staining with Wright's stain, and the value was always >95%. The macrophage suspensions were adjusted to 7 x 105 cells/ml in IS medium, and 0.1 ml ofthis preparation was seeded into flat-bottomed, 96-well microtiter plates (Corning Glass Works, Corning, NY) and cultured for three days with or without 0.1 ml ofa 1:10dilution ofdifferent SF samples or for three days with Con A-activated lymphocyte supernatants. The cultures were pulsed with 1 J.lCi of [3H]thymidine for the final 36 h. After cold shock and incubation in 0.02% EDTA plus 0.02% lidocaine (Burns VeterinarySupply, Oakland, Calit), the macrophages were released from the wells and harvested mechanically onto filter disks by using a semi-automated cell harvester (Brandell, Rockville, Md). The disks were immersed in 1 ml of PPO-POPOP (Sigma) scintillation cocktail, and ['Hlthymidine incorporation was measured by liquid scintillation spectrometry (Beckman Instruments, Irvine, Calif). Titration ofvirus. As previously described [19], CAE V was detected by adding twofold dilutions of cell-free SF to cultures of caprine synovial membrane cells and then observing these indicator cells for two weeks for CPE characteristic of CAEV infection. These titrations wereexpressed as those cultures with 50% of the replicates infected, or the TCID so Statistics. Student's t test was done to determine significant differences between means. In some instances Pearson's correlation coefficients were determined. The significance ofthe correlation coefficients was determined by Student's t test and by using' the SAS/GRAPH@ statistical software package (SAS Institute, Cary, NC)..~~.';'.0.;~.., 'lit - 4 Figure 1. Top, a Wright-stained preparation ofsf cells showing a macrophage-like cell in mitosis and surrounded by lymphocytes. Bottom, a Wright-stained preparation of SF cells in the autoradiographic assay. SF cells were allowed to adhere to plastic and incubated with [lh]thymidine and yeast, and autoradiography was performed. A cell is shown that has silver grains over its nuclei, has phagocytized nine yeast cells, is adherent, and is the size and shape of a macrophage (bars = 10 J.1m).

4 1196 Jutila and Banks Results Analysis of cells from the SF. On the basis of morphology, production of nonspecific esterase, phagocytosis, and adherence, we determined that ofsf cells from control joints were macrophages. The remaining percentage of cells consisted of neutrophils, lymphocytes, and/or fibroblast-like cells. The total cell number in control joints ranged from 104/mL to 8 X lq4/ml. In SF obtained from CAE V-infected joints, macrophages, (with the same characteristics as above), constituted anywhere from 30% to of the SF cell population. Lymphocytes were the other major cell population, with neutrophils being present occasionally. The number of cells in the infected joints ranged from 5 X 10 4/mL to 3 X 10'/mL. These results are similar to those reported by others [1,4, 5, 20]. In the MI and autoradiographic assays presented below, only cells with characteristics of macrophages were included. Figure 1 (top) is an example of an adherent macrophage in mitosis and surrounded by lymphocytes. Figure 1 (bottom) is an example of a dividing macrophage in the autoradiographic assay. The cell is adherent, has phagocytized nine yeast cells, and has the size and morphological characteristic of a macrophage. Studies of the same macrophage populations with the two assay methods showed that the MI analysis highly correlated with autoradiographic values (r =.98, P <.001), a finding indicating that the two assays are measuring macrophage division. Analysis of macrophage division. MI and in vitro autoradiographic analysis showed that there was SF macrophage division in the normal, uninfected and CAEV-infected carpus, but goats infected Table 1. with CAEV had three- to six-fold greater macrophage division in SF (table 1). Macrophage division was increased similarly in goats infected with CAEV by feeding, inoculation iv, and inoculation into the carpus. The length of time the goats were infected did not affect the results, because goats infected with CAEV for six months, 1.5 years, and four years all had similarly increased macrophage division (table 1). Division occurred in vivo in four CAEV-infected and two uninfected goats, as shown by the uptake by SF macrophages of ph]thymidine that was injected directly into the carpus. The percentage of macrophages that incorporated ph]thymidine during the 30-min in vivo labeling was ± in CAEV-infected vs ± 0.3% in uninfected goats. The division ofperipheral blood monocytes rarely occurred in either infected or uninfected goats. For three weeks, monocyte division was studied at weekly intervals by autoradiography in four infected and four uninfected goats. The mean percentage ofdividing monocytes in infected goats was 0.04 ± 0.02 and 0.06 ± 0.05 in uninfected goats. SF macrophage division, arthritis, and concentration ofinfectious virus. Experiments were done to examine the associationofincreased macrophagedivision with arthritis and the amount of infectious CAEV in the joint. Arthritis was defined as increased carpal joint swelling and an increase in the number of macrophages within the SF. When the division of SF macro phages of 58 goats was compared with the amount of joint swelling (carpus/metacarpus [C/M] ratio), we found a positive association (r =.72, P <.001) between these two parameters. This association was strongest in joints just beginning to SF macrophage division as detected by mitotic figures and autoradiography Method of analysist MI AutoR Experiment. of Duration of no.* animals/group infection (mo) Infected Uninfected Infected Uninfected I ± O.03l 0.03 ± 0.01 ND ND 2 4 I 0.30 ± ± ± ± ± ± ± ± 0.4 TE. ND = not done. Data are expressed as mean ± SE. * Goats were infected by injecting CAEV into goats in the following manner: experiment I, iv and feeding at six months of age; experiment 2, CAEV injected iv at five months of age; experiment 3, intraarticular injection at to-ii d of age. t MI is the percentage of macrophages with mitotic figures; AutoR is the percentage of macrophages that have incorporated [3Hlthymidine by techniques described in Materials and Methods. l All differences of means of the autoradiographically analyzed cells are significant (P <.01) by Student's t test. For experiments I, 2, and 3, P was <.01,.05, and.05, respectively, for means of infected vs, uninfected MIs.

5 Increased Macrophage Division in CAE 1197 develop arthritis and weakest in the more severely swollen joints. Likewise, as the numbers of macrophages in the SF increased, there was a significant increase in macrophage division (r =.70, P <.01). There were consistent examples, however, of high macrophage division without any association with inflammation in the joints. When the concentration of virus in SF was compared with SF macrophage division (table 2), a large number ofdividing SF macrophages was seen in every instance that virus could be detected. However, there was one sample from joint fluid that had large numbers of dividing macrophages but no free infectious virus. Macrophage mitogenic activity in SF. The 96- Table 2. Comparison of detectable infectious CAEV in SF and the number of dividing SF macrophages. Experiment no. * 2 3 Macrophage division MI/mLt o AutoR/mLt 57 X x x x x x x x x 103 CAEV recovered (titer /ml) Yes (10) Yes (10) Yes (10) Yes (10) Yes (10) TE. = not done. * Experiment 1 used SF samples from both carpal joints of four two-year-old goats infected by feeding them CAEV at one month of age. Experiment 2 used SF samples from the left carpal joint of four four-year-old goats infected by iv and ia inoculation of CAEV at d of age. Experiment 3 used SF samples from both carpal joints of five 1.5-y-old goats infected by iv inoculation of CAE V at six months of age. t MI/mL is the number of macrophage mitotic figures/ml of SF. t AutoR/mL is the number of macrophages/ml of SF that have incorporated [3H]thymidine, as determined by multiplying the number of macrophages/ml of SF by the percentage of dividing macrophages. The recovery of infectious CAEV from cell-free SF in the joints at the time of cell analysis, expressed as TCIO so well division assay was used to determine if SF from infected goats had mitogenic activity for goat macrophages. Division of mammary macrophages from four uninfected goats was not increased after a threeday culture with SF from either infected or uninfected goats. In some instances SF reduced division. In contrast, mammary macrophages from four goats infected with CAEV had significant increases in proliferation during culturing in the presence of SF from infected goats (table 3). One CAEV-infected goat was sampled every two to three weeks over a period of four months, and the proliferative responses ofits macrophages to SF from infected goats were consistently increased. Ifthe effect ofsf was to increase macrophage division, as suggested by these results, then we predicted that there would be an association between the SF's ability to promote macrophage division in our 96-well assay and the number of dividing macrophages in the joint that supplied the same synovial sample. Samples of SF were collected, and separated macrophages were assayed for division by ph]thymidine incorporation. The cell-free SF was placed with mammary macrophages from infected goats, and the amount of incorporation was similarly determined. Twenty-two such assays showed a positive correlation (r =.61, P <.01) between the magnitude of stimulation by SF and the number of dividing macrophages derived from the same sample of SF. To examine potential cellular sources of mitogenic activity in SF from goats infected with CAEV, we tested macrophages from infected goats for in vitro division when cultured with supernatants from Con A-stimulated lymphocytes. Macrophages from tissue from CAEV-infected goats, but not macrophages from uninfected goats, had increased division when cultured in the presence ofactivated lymphocytesupernatants, as compared with the amount of division that occurred when cultured with Con A alone, unstimulated lymphocyte supernatants, or control medium. Dialyzed, activated lymphocyte supernatants retained mitogenic activity (table 4). To determine if activation of lymphocytes in vivo resulted in greater increases in SF macrophage division in CAEV-infected goats than in uninfected goats, as seen with SF and lymphocyte supernatants in vitro, we did the following experiment. CAEVinfected and uninfected goats, previously immunized with mhsa, were injected in the left carpus with mhsa and in the right carpus with saline. Immedi-

6 1198 Jutila and Banks Table 3. Effect of SF from CAEV -infected and uninfected goats on the incorporation of [JH]thymidine by macrophages from CAEV-infected and uninfected goats. Source of macrophagest Uninfected goats Infected goats Source of SF. experiment no. * Mediuml SF Mediuml SF Uninfected goats (1.0) (1.4) (1.1) (1.4) (0.1) (1.2) (1.8) (0.6) (1.4) (1.6) 6 ND ND ND (1.1) Mean ± SE 1.1 ± ± 0.3 Infected goats (1.5) (4.1) (0.8) (3.5) (0.4) (4.2) (0.8) (5.2) (0.7) (4.1) 6 ND ND ND (3.1) 7 ND ND ND (5.0) Mean ± SE 0.8 ± ± * SF was obtained from CAEV-infected and uninfected goats and was tested at a 1:20 dilution. t Macrophages were collected from mammary glands of CAEV-infected and uninfected goats. t Mean cpm of quadruplicate wells of macrophages cultured with medium alone or SF from infected or uninfected goats. The quadruplicate samples had <20070 variation from the mean. The value in parentheses is the mean cpm with SF/the mean cpm of macrophages cultured in medium alone. II p <.001 for the mean values for uninfected vs. infected goats. ately after injecting mhsa, an acute episode of arthritis ensued, as reflected by swelling in joints (Cz'M ratio> 1.90) and influx of inflammatory cells (> 5 X losiml). The full analysis and time-course studies of the AlA in goats are contained in a separate report [20]. In one group of goats, cells from SF were obtained six days after the intraarticular injection, which occurred at the peak of the acute inflammatory response. In a second group of goats, SF cells were obtained 14 w later, during the chronic stage of arthritis. Different groups were used for the acute and chronic analysis to avoid the complications of Table 4. Effect of supernatants from lymphocyte cultures on the division of macrophages from CAEV-infected and uninfected goats. Source of macrophages Test material 50% Control lymphocyte supernatants 50% Con A lymphocyte supernatants 25% Con A lymphocyte supernatants 50% Dialyzed Con A lymphocyte supernatants 25% Dialyzed Con A lymphocyte supernatants Con A alone (1 ug/rnl.) Con A alone (0.1 ug/rnl.) Infected goats 1.1 ± ± 3.0* 4.8 ± 1.7* 6.5 ± 1.5* 4.5 ± 0.70* 0.8 ± ± 0.24 Uninfected goats 0.59 ± ± ± 0.41 ND ND ND ND te. Data are expressed as mean stimulation indices ± SE of three experiments. The stimulation indices are the average cpm of quadruplicate wells with various test materials/the average cpm of quadruplicate wells of macrophages cultured in medium alone. The cpm in medium alone ranged from 1077 to 2480 with macrophages from infected goats and from 931 fo 4650 with macrophages from uninfected goats. Each series of quadruplicate samples had <20% variation from the mean. ND = not done. * p <.001 for mean control lymphocyte supernatant vs. other test material.

7 Increased Macrophage Division in CAE 1199 Table 5. Effect of antigen-induced arthritis initiated by a nonviral antigen on SF macrophage division in CAEVinfected and uninfected goats. Acute arthritis" Chronic arthritist Macrophage division Macrophage division Joint Joint Joint injection swellingl MI MllmL swelling l MI MllmL Uninfected goats Saline 1.65 ± ± ± ± ± ± 30 Antigen 1.99 ± ± ± ± ± ± 40 Infected goats Saline 1.73 ± ± ± ± ± ± 400(} Antigen 2.12 ± ± ± ± ± ± 8000 TE. Data are expressed as mean ± SE. An immunized group of four infected and four uninfected goats was injected with antigen into the left carpus and with saline into the right, and joint cells and carpal swelling were examined six days later. t An immunized group of six infected and six uninfected goats was treated as described above, except joint determinations were conducted 14 w after the intraarticular injections. hoint swelling was determined by the carpus/metacarpus ratio, as described in Materials and Methods. MI/mL is the number of macrophage mitotic figures/ml of SF. trauma-induced inflammation that arise from multiple entries into the joint. Division of SF macrophages was assessed at the different times. In the uninfected goats, there was a significantly lower percentage of dividing macrophages at six days after antigen injection, as compared with the percentage in saline-injected control joints, a finding perhaps reflecting the immune-mediated influx of nondividing monocytes. The actual number of dividing macrophages was essentially the same in the joints ofgoats injected with either saline or antigen. After 14w there was no difference in the percentage or in the number of dividing macrophpages in the joints of goats with AlA vs. saline-injected control joints (table 5). The division ofsf macrophages in CAEV-infected goats after six days was similar to the results obtained in the uninfected goats in that the percentage of dividing macrophages decreased in goats with acute AlA as compared with the saline-injected control, but the number ofdividing macrophages in the joints receiving eithersalineor antigenremainedthesame. During this acute inflammatory reaction, however, more division occurred in the infected goats than in the uninfected goats. Fourteen weeks later, division, as reflected by MI and the number of mitotic figures, was significantly greater in the AIA-CAEV joints (table 5). At this later time the number ofmacrophages in the joint, the concentration of CAEV, and the joint swelling were higher in joints undergoing AlA, compared with joints that had been injected with saline. Joints of CAEV-infected goats undergoing AlA had greater macrophage division than did joints that were not stimulated with antigen (joints injected with saline). Thus, there was no increase in macrophage division during AlA in uninfected goats, but in infected goats, SF macrophages responded by increased division. Discussion Infecting goats with the lentivirus CAEV resulted in an enhancement ofsf macrophage division. The dividing cells were shown to be adherent, phagocytic, and positive for nonspecific esterase, and these cells had morphological characteristics typical of macrophages. The study of three groups of goats, varying in age, length of infection, and route of infection, showed a three- to sixfold increase in the percentageof dividing macrophages when compared with age-matched controls. In the overall study the division in infected animals ranged from 2070 to 16%, whereas in noninfected animals the division ranged from 1% to This difference was noted by three techniques, including one that labeled the dividing cells in the animals. In addition, there was a significant correlation between the number of cells in the joint and the amount of division, a predicative result if the laboratory results were correct. We also found that macrophages of the SF can divide at a

8 1200 Jutila and Banks rate similar to that of other tissue macrophages in other animals [25-33] and other tissues (mammary gland) of the goat [22]. Preliminary experiments were conducted to determine how viral infectionofthe goats leads to greater SF macrophage division. One explanation was an increased trafficking of dividing monoblasts to the joints of animals infected with CAEV. monocytosis occurs in CAEV-infected animals [34]t and we were unable to detect any difference in the concentration of dividing monoblasts in the peripheral blood of infected and noninfected goats. In our second attempt to explain the greater macrophage division we looked for an increased response of macrophages from infected animals to potential growth factors in SF. Tissue macrophages from CAE V-infected goats, but not from uninfected goats, proliferated in vitro in response to SF from infected goats. CAE V infection was associated with macrophage mitogenic activity in SFt and we found a positive correlation between the amount ofmacrophage division stimulated in vitro by an SF sample and the amount of macrophage division occurring in the joint that supplied the SF. Because lymphocytes are often a major constituent of the CAEV-induced lesion, they may have been the source ofthe mitogenic activity in SF. Consistent with this suggestion was our observation that phytolectin-stimulated lymphocyte supernatants contained a nondialyzable factor that enhanced the in vitro division of macro phages from CAEV-infected, but not uninfected, animals. Because the supernatants depressed the low amount of division occurring in uninfected macrophage populations, the difference between infected and uninfected animals was over 20-fold. Wethen determined if triggering lymphocytes in vivo resulted in increased macrophage division in goats infected with CAEV. Table 5 shows that an immune reaction in the joints ofcae V-infectedgoats induced threefold more dividing cells/hlo macrophages than that seen in uninfected goats. These studies show that macrophages from goats infected with CAEV have enhanced division in the presence of soluble immune factors. How infection of goats by CAEV induces these changes in macrophages would seem most directly, but not necessarily, related to viral infection of the macrophage itself. Macrophages can be infected by CAEV in vitro, a process leadingto release ofinfectious virus and the rapid appearance of CPE in almost all cultured cells [13-16]. This result does not occur in vivo. Only % of macrophages in the SF ofinfected animals have viral antigen detectable by immunofluorescence, and this antigen is only detectable during the first four weeks of infection [I]. The site ofviral infection after one month ofinfection is still unknown. Search for CAE V-infectedcells by in situ hybridization has not been reported, although this technique has been used to study the closely related lentivirus of sheep, visna. By inoculation with large amounts ofthis virus, the visna virus can be detected by in situ hybridization in a minority of alveolar and CSF macrophages [13, 35, 36] within the first few days of experimental infection. These cells have very restricted viral replication, and the number ofdemonstrable infected cells after a few weeks of infection is < [37]. Because we do not know if macrophages are infected, we cannot presently resolve the role of virus in increasing macrophage division; however, our observations suggest two possible relations between viral infection and macrophage division. First, those joints with detectable CAEV had the greatest number ofdividing macrophages, and when enhanced macrophage division was triggered by an immune response in an infected joint, slightly more virus was produced. Others have proposed that there is a connection beteen macrophage division in bone marrow and the dissemination of visna virus [13] and that there is an association between dividing macro phages and avian [17] and feline [18] retrovirus infections. Second, CAEV macrophage infection may increase receptors for mitogenic factors, and this increase would explain the greater in vitro division of macrophages ofinfected goats when cultured with mitogenic SF and activated lymphocyte supernatants. This mechanism, if correct, would parallel the finding that the human T lymphocyte virus type I infection of T cells causes greater expression of the interleukin-2 receptor [38, 39]. We found that the amount of SF macrophage division was correlated with the degree ofarthritis and SF macrophage number. The increased division may contribute to the lesions of CAEV infection by increasing the number of macrophages that replace normal tissue [1-3, 5]. Once more macrophages are present in the joint, these cells may releasetheir secretory products [40], an action attracting [41] and stimulating the growth of more cells [42, 43] or directly damaging articular surfaces [44]. In addition, increased la antigen expression of macrophages from animals infected with CAEV [45], the aug-

9 Increased Macrophage Division in CAE 1201 mented blastogenesis of lymphocytes that occurs in CAEV-infected goats [34, 36], and the finding here of greater response of macrophages to lymphocyte products suggest that CAEV infection alters the immune system so that upon entry of any antigen, excessive reactions occur. Indeed, this suggestion has been partially proved by experiments that showed more progressive AlA in goats infected with CAEV than in uninfected goats [20]. Therefore, we believe that the pathogenesis of the arthritis is modified by the effect of the virus on macrophages and the response ofmacrophages to lymphocyte products. Any stimulation oflymphocytes that results in release of macrophagegrowth factor would be followed by another cycle of macrophage division, which would exacerbate the lesion. This study has uncovered a novel effect of a. nononcogenic retrovirus on cellular function, and this effect might be relevant to other lentivirus infections. A common thread linking the goat, sheep, and horse lentiviruses is the macrophage and aberrant macrophage functions [45, 47, 48]. HIV is the first-described human lentivirus [8-10] that also infects macrophages [49-51]. It is intriguing that some patients with AIDS have tumor-like proliferation of lymph node macrophages [52] and the development of brain macrophage nodules [53, 54]. If HIV induces greater macrophage division, as with CAEV, the results of our experiments suggest that immune responses could lead to an increase in these lesions and possibly to expression and expansion of the virus in the macrophage population, an action possibly contributing to the immune dysfunction and infection of lymphocytes. References 1. Adams DS, Crawford TB, Klevjer-Anderson P. A pathogenetic study ofthe early connective tissue lesions ofviral caprine arthritis-encephalitis. Am J Pathol 1980;99: Cork LC, Hadlow WJ, Crawford TB, Gorham JR, Piper RC. Infectious leukoencephalomyelitis of young goats. 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