Received 21 January 1997/Accepted 25 April 1997

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1997, p Vol. 71, No X/97/$ Copyright 1997, American Society for Microbiology Type 1 and Type 2 Cytokine Gene Expression by Viral gp135 Surface Protein-Activated T Lymphocytes in Caprine Arthritis-Encephalitis Lentivirus Infection W. P. CHEEVERS, 1 * J. C. BEYER, 1 AND D. P. KNOWLES 1,2 Department of Veterinary Microbiology and Pathology, Washington State University, 1 and Animal Diseases Research Unit, U.S. Department of Agriculture-Agricultural Research Service, 2 Pullman, Washington Received 21 January 1997/Accepted 25 April 1997 Peripheral blood mononuclear cells (PBMC) from Saanen goats experimentally infected with the lentivirus caprine arthritis-encephalitis virus (CAEV) were evaluated by semiquantitative reverse transcriptase PCR for gamma interferon (IFN- ), interleukin-4 (IL-4), and IL-2 gene expression following in vitro stimulation with purified CAEV gp135 surface protein (SU). Studies examined three goats with chronic arthritis and four clinically asymptomatic goats at 5 years postinfection. SU-responsive IFN- mrna-positive cells and IL-4 mrna-positive cells in PBMC from infected goats reflected differences in lymphokine balance associated with disease status. IFN- mrna-positive cells were dominant in PBMC from asymptomatic goats, whereas SU-responsive IL-4 mrna-positive cells were dominant in PBMC from goats with arthritis. IL-2 gene expression was not responsive to SU stimulation of PBMC from either asymptomatic or arthritic goats. Lymphokine mrna profiles in SU-stimulated PBMC were dependent on the presence of CD4 T lymphocytes. The results indicate that asymptomatic goats have a dominant population of CAEV SU-reactive T-helper 1 (Th1)-like lymphocytes in PBMC whereas goats with clinical arthritis have a dominant population of SU-reactive Th2-like lymphocytes. Caprine arthritis-encephalitis virus (CAEV) is a lentivirus of domestic goats that infects monocytes and macrophages and causes chronic inflammatory disease, primarily arthritis or mastitis (8, 18, 30). Like human immunodeficiency virus (HIV) infection, goats infected with CAEV do not uniformly progress to clinically apparent disease. Many naturally or experimentally infected goats are long-term nonprogressors (LTNP) characterized by lifelong persistent infection, relatively low virus loads, low anti-caev antibody titers, and lack of significant lesion development (2, 6, 7, 22, 32, 36). In contrast, up to 40% of infected goats develop chronic arthritis (6, 7, 13, 16) characterized by infiltration of the synovium by B lymphocytes and plasma cells, major histocompatibility complex class II activated CD4 and CD8 T lymphocytes, and activated macrophages (6, 36). CAEV-induced arthritis follows a progressive course with clinical signs of periarticular swelling, excessive synovial fluid containing mononuclear inflammatory cells, and radiographic changes involving soft-tissue mineralization and erosion of articular surfaces (6, 7, 37). Compared to LTNP goats, arthritic goats have higher virus loads in synovial tissue and increased serum and synovial fluid titers of polyclonal immunoglobulin G1 and CAEV-reactive antibodies, particularly to surface (SU) and transmembrane envelope glycoproteins (2, 3, 6, 21, 22, 25, 32). Arthritic goats also develop neutralizing antibodies to CAEV SU epitopes (7, 23, 26); however, neutralizing antibody fails to regulate CAEV replication in arthritic goats and is undetectable in most LTNP goats in which virus expression is relatively restricted (7, 9). These findings may be interpreted in terms of CAEV SUreactive T-helper 1 (Th1) and Th2 lymphocytes, which are * Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA Phone: (509) Fax: (509) cheevers@vetmed.wsu.edu. defined in mice and humans by differential lymphokine production and immune effector functions: terminally differentiated Th1 cells, the primary effectors of cell-mediated immune function, are characterized by secretion of gamma interferon (IFN- ) and interleukin-2 (IL-2), whereas Th2 lymphocytes are characterized by secretion of IL-4 and other cytokines that promote B-cell differentiation (1). Thus, we have suggested that differential activation of CAEV-reactive Th subsets may influence both the extent of virus expression and the clinical outcome of CAEV infection (32, 36). In the present study, one aspect of this hypothesis was examined by analysis of type 1 and type 2 lymphokine gene expression in circulating CAEV SU-responsive T lymphocytes from LTNP and arthritic goats. Goats used in this study were from a CAEV-free Saanen breeding herd maintained at Washington State University. Newborn kids were infected intravenously with % tissue culture infective doses of biologically cloned CAEV-63 (14) or CAEV-Co (29). All infected goats seroconverted for antibodies to CAEV structural proteins by 4 months postinfection (p.i.) and were seropositive and PCR positive for CAEV gag in peripheral blood mononuclear cell (PBMC) DNA at 5 years (p.i.) (data not shown). Clinical arthritis was evaluated in terms of periarticular swelling of carpal joints as monitored by the carpal/metacarpal (C/MC) ratio (6, 7). At the time of experiments reported here (59 to 66 months p.i.), the mean C/MC ratio the standard deviation for five age-matched uninfected goats was The clinical status of infected goats was as follows: two CAEV-63-infected goats (91-11 and 91-19) and one CAEV-Co-infected goat (91-27) had bilateral arthritis, as indicated by a mean C/MC ratio of (6), whereas asymptomatic CAEV-63-infected goats 91-5, 91-9, and and CAEV-Co-infected goat had mean C/MC ratios in the normal range ( ). PBMC prepared by density gradient centrifugation were cultured in six-well plates at cells/well in RPMI 1640 medium with 10% inactivated fetal calf serum (32). The plates 6259

2 6260 NOTES J. VIROL. TABLE 1. Primers for semiquantitative RT-PCR analysis of cytokine mrna Cytokine Primer Positions Size of PCR product (bp) IFN- a Forward: 5 -TAGCTAAGGGTGGGCCTCTTTTCTCA Reverse: 5 -TGCAGGCAGGAGAACCATTACATTGA IL-4 a Forward: 5 -TAGCTTCTCCTGATAAACTA 1 20 Reverse: 5 -ATGAGTTATAAATATATAAATA IL-2 a Forward: 5 -ATGTACCAGATACCACTCTTGTCTT Reverse: 5 -TCAAGTCATTGTTGAGTAGAT Actin b Forward: 5 -ACCAACTGGGACGACATGGAG Reverse: 5 -GCATTTGCGGTGGACAATGGA 890 a Primers for these cytokines were derived from the nucleotide sequence of caprine cytokine cdna clones (4). b Primers for this cytokine were derived from the nucleotide sequence of bovine -actin cdna (12). were incubated at 37 C in humidified CO 2 for 144 h with or without CAEV gp135 SU, purified by affinity chromatography on CNBr-activated Sepharose 4B (Pharmacia) coupled to anti-su monoclonal antibody (MAb) F7-299 (32). For semiquantitative reverse transcriptase (RT)-PCR, PBMC RNA was extracted with TRIzol reagent (Gibco BRL), and lymphokine mrna levels were expressed as a function of the amount of -actin mrna for each RNA sample (12). The sequences of primers used for RT-PCR reactions are shown in Table 1. For RT reactions, a GeneAmp RNA PCR kit (Perkin- Elmer) was used with reverse primers for caprine IFN-, IL-4, IL-2, or -actin mrna. Equivalent amounts of lymphokine or -actin cdna were then amplified in separate PCR reactions using PCR Master (Boehringer Mannheim) with 1.5 mm MgCl 2 for IFN-, IL-2, and -actin cdna amplification or 5 mm MgCl 2 for IL-4 cdna amplification. PCR consisted of 35 cycles, with denaturation, annealing, and extension conditions of 94 C (15 s), 50 C (58 s), and 72 C (71 s), in a Perkin- Elmer thermocycler (9600 GeneAmp PCR System). PCR products were resolved by electrophoresis in 1% agarose gels (SeaKem MR; FMC BioProducts) in 0.04 M Tris-acetate (ph 8.5) M EDTA containing 0.05 g of ethidium bromide per ml. The UV fluorescence of cdna bands was measured with an IS-1000 Digital Imaging System (Alpha Innotec). Fluorescence values for lymphokine mrna RT-PCR products were corrected to enable direct comparison with the measured fluorescence of the 890-bp RT-PCR product of -actin mrna (33). Correction values were 890/385 (2.31) for IFN-, 890/535 (1.66) for IL-4, and 890/468 (1.90) for IL-2 (33). Data are presented as follows: corrected fluorescence of lymphokine cdna/measured fluorescence of -actin cdna. We initially examined IFN- and IL-4 gene expression in CAEV SU-stimulated PBMC from selected CAEV-63- and CAEV-Co-infected goats at 59 months p.i. This experiment included CAEV-63-infected LTNP goat 91-5 and arthritic goat and CAEV-Co-infected LTNP goat and arthritic goat SU-stimulated PBMC from an age-matched uninfected goat (91-36c) was included as a control. Figure 1A shows ethidium bromide-stained RT-PCR products of IFN- mrna (lanes 1, 3, 5, 7, and 9), IL-4 mrna (lanes 2, 4, 6, 8, and 10), and -actin mrna (lanes 11 to 15) resolved by agarose gel electrophoresis. Lymphokine cdna/ -actin cdna ratios derived from this gel are shown in Fig. 1B. These results demonstrated that CAEV SU-responsive IFN- and IL-4 gene expression in PBMC reflected differences in lymphokine balance associated with disease status. IFN- gene expression was preferentially enhanced relative to IL-4 gene expression in SU-stimulated PBMC of LTNP goats. A substantial level of IFN- gene expression was also present in SU-stimulated PBMC of goats with arthritis; however, IL-4 gene expression was enhanced by SU stimulation of arthritic-goat PBMC compared to that of SU-stimulated PBMC of either uninfected or LTNP goats. The initial virus inoculum (CAEV-63 or CAEV- Co) did not affect these lymphokine mrna profiles. Additional studies evaluated IFN- and IL-4 gene expression in unstimulated and CAEV SU-stimulated PBMC from uninfected goat 91-7c, LTNP goats 91-5 and 91-9, and arthritic goat at 59 months p.i. (Fig. 2A). SU-stimulated PBMC from LTNP goats 91-5, 91-10, and and arthritic goats 91-11, 91-19, and were also examined at 62 months p.i. (Fig. 2B). These data confirm and extend the results shown in Fig. 1 and support the following conclusions: (i) the relatively low IFN- and IL-4 mrna levels in unstimulated PBMC of uninfected goat 91-7c were not enhanced by in vitro stimulation with SU (Fig. 2A), indicating the absence of SU-respon- FIG. 1. Semiquantitative RT-PCR analysis of IFN- and IL-4 mrna in CAEV SU-stimulated PBMC from goats infected with CAEV-63 or CAEV-Co. RT-PCR reactions for IFN-, IL-4, and -actin mrna used 774 ng of RNA (determined by A 260 ) extracted from PBMC obtained at 59 months p.i. and cultured 144 h with 3 g of affinity-purified CAEV SU per ml. (A) Separation of RT-PCR products by agarose gel electrophoresis. IFN-, PCR amplification of a 385-bp fragment of caprine IFN- cdna clone; IL-4, PCR amplification of a 535-bp fragment of caprine IL-4 cdna. Lanes: 1, 3, 5, 7, and 9, RT-PCR products of IFN- mrna; 2, 4, 6, 8, and 10, RT-PCR products of IL-4 mrna; 11 to 15, RT-PCR products of -actin mrna of SU-stimulated PBMC from goats 36c, 5, 32, 11, and 27. (B) Corrected fluorescence of lymphokine mrna RT-PCR products/measured fluorescence of -actin mrna RT-PCR products obtained by digital image analysis of the ethidium bromide-stained gel shown in panel A.

3 VOL. 71, 1997 NOTES 6261 FIG. 2. Semiquantitative RT-PCR analysis of IFN- and IL-4 mrna in unstimulated and CAEV SU-stimulated PBMC from uninfected and CAEV-infected goats. Data with error bars represent the means standard deviations of two independent RT-PCR analyses using the same RNA. (A) RT-PCR reactions utilizing 726 ng of RNA extracted from PBMC obtained at 59 months p.i. and cultured 144 h with ( ) or without ( ) 3 g of CAEV SU per ml. (B) RT-PCR reactions utilizing 318 ng of RNA extracted from PBMC obtained at 62 months p.i. and cultured 144 h with CAEV SU. Goat numbers are shown along the x axis. sive cells in uninfected-goat PBMC; (ii) expression of IFN- and IL-4 mrna was not enhanced in infected-goat PBMC cultured without SU (Fig. 2A); (iii) IFN- gene expression was markedly enhanced in SU-stimulated PBMC of all LTNP and arthritic goats (Fig. 2A and B); (iv) IL-4 gene expression in SU-stimulated PBMC of LTNP goats was variable, with goats 91-5 and 91-9 exhibiting enhancement but goats and showing little or no enhancement (Fig. 2A and B), while IL-4 gene expression was markedly enhanced in SU-stimulated PBMC from all goats with arthritis (Fig. 2A and B). The results of semiquantitative RT-PCR analyses of IL-2 mrna levels are shown in Fig. 3. In contrast to IFN- and IL-4, IL-2 gene expression was not enhanced in CAEV SUstimulated PBMC compared to unstimulated PBMC. Lymphokine mrna profiles in CAEV SU-stimulated PBMC were not due to differences in the number of potentially responsive lymphocytes or potential antigen-presenting cells in PBMC cultures; flow cytometry using MAbs to caprine differentiation markers as previously described (32, 36, 37) confirmed that PBMC from all goats at 59 months p.i. had similar proportions of CD4 T lymphocytes (31.3% 2.4%), CD8 T lymphocytes (17.8% 3.7%), B lymphocytes (14.5% 3.7%), and monocytes (3.5% 0.6%) at the time of culture. To identify CAEV SU-responsive cells, PBMC from goats 91-5 and at 66 months p.i. were evaluated for SU-induced lymphokine gene expression following depletion of CD4 T lymphocytes. Similar to previous results (32), complementmediated lysis of cells binding anti-cd4 MAb GC50A1 (15) depleted 92% of CD4 T lymphocytes in goat 91-5 PBMC and 97% of CD4 T lymphocytes in goat PBMC without affecting CD8 cells, -positive cells, B cells, or monocytes (data not shown). As expected, SU-responsive IFN- mrnapositive cells were dominant in undepleted PBMC from LTNP goat 91-5 whereas SU-responsive IL-4 mrna-positive cells were increased in undepleted PBMC from arthritic goat (Fig. 4). Selective depletion of CD4 T lymphocytes reduced SU-responsive IFN- and IL-2 gene expression and eliminated SU-responsive IL-4 gene expression (Fig. 4). In summary, this study demonstrates that CAEV-infected LTNP goats have circulating T lymphocytes that exhibit predominant IFN- gene expression in response to stimulation with CAEV SU whereas goats with clinical arthritis have increased numbers of SU-responsive IL-4 mrna-positive T lymphocytes. A PBMC depletion experiment identified SUresponsive cells as CD4 T lymphocytes. An important aspect of these results is that CAEV SU-reactive Th lymphocytes exclusively expressing IFN- or IL-4 mrna were not detected in unfractionated PBMC, even when selected for a specific recall response by stimulation with purified antigen. In addition, relatively high spontaneous levels of IL-2 mrna in unstimulated PBMC prevented identification of this lymphokine marker of Th1 cells in CAEV SU-stimulated PBMC. Therefore, the data do not permit a statement of the actual proportions or degree of differentiation of SU-responsive Th lymphocyte phenotypes. Flow cytometry experiments using anti- FIG. 3. Semiquantitative RT-PCR analysis of IL-2 mrna in unstimulated and CAEV SU-stimulated PBMC from uninfected and CAEV-infected goats. RT-PCR analyses used (i) 726-ng quantities of RNA from PBMC of goats 7c, 5, 9, and 11 obtained at 59 months p.i. and cultured with ( ) or without ( ) CAEV SU and (ii) 318-ng quantities of RNA from PBMC of goats 36c, 5, 9, 10, 32, 11, 19, and 27 obtained at 62 months p.i. and cultured with or without CAEV SU. Data represent the means standard deviations of IL-2 cdna/ -actin cdna values.

4 6262 NOTES J. VIROL. FIG. 4. Semiquantitative RT-PCR analysis of lymphokine mrna in CAEV SU-stimulated PBMC following depletion of CD4 T lymphocytes. RT-PCR reactions used 500 ng of RNA from PBMC of goats 91-5 and obtained at 66 months p.i. and cultured with CAEV SU with or without depletion of CD4 T lymphocytes. caprine lymphokine MAbs to detect lymphokines produced by individual T cells will be necessary to identify terminally differentiated Th1 and Th2 lymphocytes as well as Th0 lymphocytes (17), which may represent intermediate cells in the differentiation of Th subsets (28). Despite these uncertainties, it is apparent that viral SUreactive Th1-like cells are dominant in PBMC of CAEV-infected LTNP goats. IFN- mrna-positive Th1-like cells are not reduced in goats with clinical arthritis compared to LTNP goats; however, SU-reactive IL-4 mrna-positive Th2-like cells are markedly increased in PBMC of arthritic goats. These results provide a mechanistic framework for previous studies of antiviral immune reactivity in relation to disease status in CAEV infection. Thus, viral SU-responsive Th1-like cells that promote a dominant cell-mediated immune response are associated with LTNP status characterized by relatively low anti-su antibody titers, vigorous Th cell proliferative responses to SU, and restricted virus expression (2, 6, 22, 32, 36). In contrast, SU-responsive Th2-like cells that promote B-cell expansion are associated with B-cell and plasma cell infiltration of joint tissue, relatively high anti-su antibody titers, depressed Th cell proliferative responses to SU, and diminished control of virus replication (2, 6, 9, 32, 36). Numerous studies of HIV infection have also correlated low virus loads and LTNP status with dominant type 1 cell-mediated immunity (5, 11, 20, 31, 35). However, studies to define cytokine mediators in anti-hiv immune responses have been inconsistent. In particular, the hypothesis that AIDS progression is associated with a shift from Th1 to Th2 dominance (10) has been difficult to confirm (19, 24, 27). However, a recent study utilizing MAbs to evaluate cytokine production by individual T cells indicated that the apparent change in cytokine balance associated with AIDS progression is the result of Th1 depletion (27), which may be due to apoptosis mediated by type 2 cytokines (11). In contrast, our results indicate that differences in lymphokine balance associated with disease status in CAEV infection are explained by a relative dominance of viral SU-responsive IL-4-positive Th lymphocytes in PBMC of arthritic goats without apparent loss of IFN- -positive Th lymphocytes. This does not necessarily contradict the HIV results (11, 27), since lymphopenia is not a detectable clinical component of CAEV-induced arthritis (37). Th subset distribution in relation to disease status in CAEV infection can be explained by at least two fundamentally different immunologic mechanisms. A shift from Th1 to Th2 dominance may promote disease progression in LTNP goats. However, we have not observed development of clinical arthritis after approximately 2 years p.i. in LTNP goats for the present study or in previous groups of experimentally infected goats monitored for up to 9 years (6, 7, 32, 36, 37). Thus, development of arthritis may not involve a shift in committed immune responses characterizing the LTNP status but rather may be dependent on preclinical dominance of Th2 responses to CAEV. A previous observation supporting early dominance of anti-caev type 2 immune responses as opposed to a Th phenotype shift in disease progression is that increased serum immunoglobulin G titers to CAEV SU at 3 months p.i. predicted the development of clinical arthritis at 12 to 15 months p.i. (32). 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