Pulmonary disease is a major problem for the 33 million individuals who are infected with HIV worldwide. Pathological changes associated with HIV-related pneumonia frequently include interstitial inflammatory exudates, formed from mononuclear cells, macrophages, and, in many cases, multinucleated giant cells [1,2]. These changes usually accompany enhanced virus replication in the macrophage population and proliferation of opportunistic pathogens such as Pneumocystis carinii (also known as Pneumocystis jiroveci), cytomegalovirus, and Mycobacterium tuberculosis[3–5]. This disease complex, resulting from replication of multiple pathogenic agents in the lungs, has also been observed in macaques infected with SIV and the simian–human immunodeficiency virus (SHIV) . Tissues from these animals thus provides a valuable resource material in which to investigate the role that cytokines, chemokines, and growth factors play in the pathogenesis of the syndrome.
Cells of macrophage lineage contribute to the pathogenesis of HIV-1 infection throughout the course of the disease . Specifically, macrophages have been shown to be the target cells for productive virus replication in the lungs of SIV/SHIV-infected macaques [6,8]. Further, there is evidence of susceptibility of alveolar macrophages to HIV-1 infection in cell culture [9,10]. Following infection with the virus, macrophages display impaired effector functions, including of phagocytosis  and of cytokine production . Using the SHIV/rhesus macaque model, we have previously demonstrated that lung pathology in SHIV-infected macaques was tightly coupled with overexpression of the chemokine monocyte chemoattractant protein 1 (MCP-1), a classic macrophage recruitment chemokine implicated in HIV disease . The rationale for assessment of expression of platelet-derived growth factor (PDGF) B chain (PDGF-B) in the lungs of SHIV-infected macaques with pulmonary disease in this study was twofold: SHIV infection in monocyte-derived macrophages (MDM) has been shown to be associated with upregulation of PDGF-B expression , and PDGF is known to be an inducer of MCP-1.
PDGF is a potent mitogenic growth factor, is frequently overexpressed in chronic pneumopathies, and is produced mainly by alveolar macrophages participating in these lesions [14–17]. It occurs as a family of disulfide-bonded dimeric isoforms (four are known: A–D) made up of pairs of PDGF chains (A, B, C) bound as homo- or heterodimers (AA,BB, CC, AB). The various forms of PDGF are produced by different types of cell and exert their cellular effects by differential binding to two specific cell membrane receptor tyrosine kinases, designated α and β [18,19]. Upon binding, they induce receptor dimerization, phosphorylation, and activation of signal transduction cascades . The PDGF α-receptor (PDGF-Rα) binds to all three PDGF chains, whereas the β-receptor (PDGF-Rβ) binds only to PDGF-B. Since PDGF has so far not been implicated in the pathogenesis of SHIV-related pneumonia, we hypothesized that regulation of PDGF expression is critical for recruitment of macrophages in the lungs, ultimately leading to the development of associated pulmonary disease.
The present study explores the association of PDGF-B chain and its cognate receptors in virus replication in the lungs of macaques with SHIV-related pneumonia.
Histopathological evaluation of lung tissues
Animals used in these studies were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and all studies were reviewed and approved by the Kansas University Medical Center's Institutional Animal Care and Use Committee. Archival lung tissues from 10 rhesus macaques infected with SHIV (SHIV89.6P or SHIVKU-2), without lung disease (four animals: RHT5, ROQ5, PHP, RJV5) and with lung disease (six animals: (PJX, RMT5, RHU5, PWJ, 49P1, 57W) were used in this study. All of the infected macaques developed AIDS-defining illnesses and were euthanized using predetermined clinical criteria, apart from PHP, which did not develop either AIDS or pulmonary disease. Details of viral inoculation, history of disease course, processing of tissue samples, and histological analyses of the tissues have been described earlier . Samples of lung tissue obtained from the apical, cardiac, and diaphragmatic lobes of each lung were fixed in 4% paraformaldehyde. Paraffin-embedded sections of lungs were stained with hematoxylin and eosin for morphological evaluation. Grocott and acid-fast special stains were performed for the evaluation of pneumocystis, fungal and acid-fast organisms. Additionally, Masson's trichrome stain was used to evaluate the extent of interstitial fibrosis.
Immunohistochemical analyses of SHIV-pneumonic lungs
Immunohistochemical analyses were carried out on paraffin-embedded sections of lung or zinc–formalin-fixed alveolar macrophages as previously described , with primary antibodies including CD3 (T cell marker; Dako Corporation, Carpinteria, California, USA), CD20 (B cell marker; Ventana Medical Systems, Tucson, Arizona, USA), HAM56 (macrophage marker; Dako), SIV p27 mouse monoclonal antibody (Advanced Biotechnologies, Columbia, Massachusetts, USA) PDGF-BB (PGF007, Mochida, Tokyo, Japan), PDGF-Rβ (Upstate, Charlottesville, Virginia, USA) and PDGF-Rα (Santa Cruz Biotechnology, Santa Cruz, California, USA).
For double-immunofluorescence staining, sections were first treated with the primary antibody, followed by treatment with Alexa Fluor 594/488-conjugated secondary antibody. The sections were then stained with another primary antibody (different species from the first primary antibody), followed by treating with Alexa Fluor 488/594-conjugated secondary antibody. After the final washing, the slides were mounted in SlowFade anti-fade reagent with 4,6-diamidino-2-phenylindole (Molecular Probes, Eugene, Origon, USA) and images were captured using a Zeiss LSM510 confocal microscope. Control slides consisted of (a) mounted tissue sections without secondary antibodies for autofluorescence; (b) red primary plus red and green secondary to check for bleed-through into green channel; (c) green primary plus green and red secondary to check for bleed-through into red channel; and (d) red and green secondary antibodies only to check for non-specific binding.
Quantification of immunohistochemical staining in the lung
Ten fields were examined on each slide and a predominant staining intensity was used for scoring. Cytoplasmic and/or cell membrane staining was semiquantitatively scored for a given cell type as 0 (absent), 0.5+ (sporadic or weak reaction of single cells), 1+ (moderate staining pattern with < 10% positive cells), 1.5+ (moderate staining pattern with 10–25% positive cells), 2+ (moderate staining pattern of 26–50% positive cells), 2.5+ (moderate staining pattern of 51–75% positive cells), or 3+ (strong staining of > 75% positive cells).
Fluorescence-activation cell sorting analysis
MDM cultures were developed and inoculated with SHIV 89.6P as described earlier . Uninfected or infected MDM at days 5 and 10 post-infection were washed twice with Hank's balanced salt solution, followed by incubation with 1 ml of Gibco trypsin–EDTA solution (0.05% trypsin; 0.53 mmol/l sodium ethylenediaminetetracetate; Invitrogen Corporation, Carlsbad, California, USA) at 37°C for 10 min. Cells were then labeled with primary PDGF-Rα (Upstate) or PDGF-Rβ (R&D Systems, Minneapolis, Minnesota, USA) antibodies on ice in the dark for 1 h and washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). Fluoroscein isothiocyanate-conjugated donkey anti-rabbit secondary antibody or donkey anti-goat secondary antibody was then used to detect the primary antibody (PDGF-Rα/β). After washing twice, the cells were fixed with 500 μl of 2% formalin and analyzed by flow cytometry. MDM were first identified by characteristic forward- and side-scatter parameters on unstained MDM, and the population was confirmed by staining with primary anti-human CD14 (Beckman-Coulter, Miami, Florida, USA). The results were recorded as mean fluorescence intensity and as percentage of positive staining population. Negative control cells were stained with isotype control primary conjugated antibodies. Samples were prepared and analyzed in duplicate, and a minimum of 10 000 cells was counted for each sample.
Migration assays were performed in a Disposable ChemoTX microplate (NeuroProbe, Cabin John, Maryland, USA) with a pore size of 8 μm. Different concentrations of PDGF-BB in PBS–1% BSA were added in each of the lower compartments and 5 × 106 MDM from SHIV-infected (4 days post-infection) or uninfected animals resuspended in 50 μl RPMI 1640 were added to the top chamber. The microplate was then incubated at 37°C with 5% CO2 for 2 h before the cells in the upper chamber were removed by washing with PBS. Cells that had migrated to the lower chamber were counted by using Cell Titer 96 Aqueous One Solution Assay (Promega, Madison, Wisconsin, USA). All assays were performed in triplicate. The migration index was calculated as the number of cells migrating toward the PDGF-BB divided by the number of cells migrating toward PBS–1% BSA only.
Real-time reverse transcriptase polymerase chain reaction
A real-time reverse transcriptase polymerase chain reaction (RT-PCR) with the SYBR Green detection method  was used for quantitative analysis of mRNA for PDGF-B, PDGF-Rα and PDGF-Rβ in the lungs. Since 97% of the sequence was found to be conserved between rhesus and human PDGF chains, the amplification of PDGF was based on human primer sequence. A RT2 PCR primer pair set (SuperArray Bioscience, Frederick, Maryland, USA) was used. Total RNA isolated from lung homogenates by trizol was then converted into first-strand complementary DNA, the template for the PCR using the Reaction Ready First Strand cDNA Synthesis Kit (SuperArray). Amplification was performed with the following PCR cycle sequence: 15 min at 95°C (stage 1), 30 s at 95°C, 30 s at 55°C followed by 30 s at 72°C (stage 2; repeated for 40 cycles), finally 72°C for 5 min (stage 3). Detection was performed with an ABI Prism 7700 sequence detector (ABI, Foster City, California, USA). Data were normalized using Ct (defined as the threshold cycle of PCR at which the amplified product is first detected) values for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample (ABI). The replication efficiencies of PDGF-B or the two receptors and the housekeeping gene, GAPDH were found to be similar. In order to calculate relative amounts of PDGF-B or PDGF-R, the average Ct value of the GAPDH was subtracted from that for each target gene to provide changes in Ct (dCt) value. The fold-change in gene expression (differences in dCt, or ddCt) was then determined as log2 relative units.
Comparisons of fold changes in PDGF (ligand and receptor) between disease-free and diseased animals were conducted using the Wilcoxon rank-sum test. Exact two-sided P values were calculated using SAS 9.1 software (SAS Institute, Cary, North Carolina, USA). Chemotaxis experiments were performed in triplicate and were repeated with rhesus MDM from at least three different animals. Data were analyzed by using t-tests or one-way ANOVA (independent group analysis). A type I error rate of 5% was used for determining statistical significance.
Lung tissues from 10 SHIV-infected rhesus macaques were evaluated in this study. Of the 10 infected animals, six (PJX, RMT5, RHU5, PWJ, 49P1, 57W) had a variety of pathological changes in the lungs (Table 1), while the other four infected animals demonstrated negligible changes. Gross evaluation of the lungs during necropsy revealed patchy to diffuse consolidation of lung parenchyma in infected animals with pulmonary disease. Cut surfaces showed patchy to uniform pale tan discoloration. All of the macaques with pulmonary disease had evidence of increased numbers of macrophages (Fig. 1a) and T cells (Fig. 1b) in the lungs, with no significant changes in B cell numbers (Table 1). Microscopic evaluation of the diseased lungs showed a variety of lesions that included histopathological changes caused by P. carinii pneumonia (PCP) in two (RMT5 and RHU5) of the six macaques (Table 1). Figure 1c includes a section of lung from macaque RMT5 showing diffuse alveolar accumulations of pink flocculent material typical of PCP, which was confirmed by Grogott silver staining (Fig. 1d).
Out of the remaining four macaques with pneumonia, three (PJX, 49P1 and 57W) had diffuse chronic interstitial pneumonia with well-formed granulomas, and prominent multinucleated giant cells (Fig. 1e,f). Interstitial parenchymal fibrosis, highlighted by the trichrome stain, was prominent in two of these macaques (PJX and 49P1; Fig. 1h). The fourth diseased animal (PWJ) had patchy acute interstitial pneumonia (diffuse alveolar damage), with intra-alveolar hyaline membranes but with no evidence of increase in parenchymal fibrosis (Table 1). Mycobacterial infections were absent in all 10 macaques as evidenced by acid-fast staining (Table 1). Additionally, there was no evidence of viral inclusions, pulmonary hypertension or malignancy in any of the animals studied.
Upregulated expression of platelet-derived growth factor B chain in SHIV-pneumonic lungs
Since PDGF is a mitogen for mesenchymal cells and is a known inducer of MCP-1, a critical chemokine involved in HIV-1 pathogenesis , the expression of PDGF-B was assessed in the lungs of macaques with and without lung disease using the real-time RT-PCR assay. As shown in Fig. 2a, all six of the SHIV-infected macaques with pulmonary disease had increased levels of PDGF-B mRNA compared with the infected animals without lung disease. The difference in the levels of PDGF-B mRNA between diseased and disease-free animals was statistically significant (P = 0.03). Interestingly, PDGF-B expression in the lungs of macaques with interstitial fibrosis (PJX, 49P1) was higher than that in the lungs of infected macaques with pulmonary disease but without fibrosis (Fig. 2a).
Based on the above findings and our earlier findings that PDGF-B expression in macrophages could be modulated by SHIV infection , virus-infected macrophages were examined to see if these cells did produce PDGF-B in the affected lungs. Confocal microscopy of lung sections using antibodies specific for macrophage marker and virus protein demonstrated that the majority of virus-infected cells in the affected lungs were macrophages (Fig. 2b). To confirm whether virus-infected cells were also producing PDGF-B, the lung sections were dual labeled with PDGF-B and viral p27-specific antibodies. As shown in Fig. 2b, most of the virus-infected cells were also positive for PDGF-B. Multinucleated giant cells present in affected lungs also showed PDGF-B protein. It should be pointed out, however, that macrophages/multinucleated giant cells were not the only source of PDGF-B in the lungs since the factor was also produced by other cells (Fig. 2b).
Further confirmation that infected macrophages were also a source of PDGF-B was achieved by staining alveolar macrophages from macaques with pulmonary disease with the PDGF-B antibody. Many of these cells expressed both PDGF-B and viral p27 (data not shown).
Upregulated expression of platelet-derived growth factor receptors in SHIV-pneumonic lungs
Real time RT-PCR using RNA extracted from lung tissues showed a statistically significant increase in both PDGF receptors in lungs from infected animals with disease compared with lungs from infected animals without pulmonary disease (Fig. 3a; exact two-sided P values comparing fold changes in diseased and disease-free animals: P = 0.0190 for PDGF-Rα; P = 0.0333 for PDGF-Rβ).
Immunostaining of sections of lung and also alveolar macrophages from the lungs of infected diseased animals showed an increase in the production of PDGF-Rα in animals with pneumonia compared with ones without (Fig. 3b). Similar findings were observed for PDGF-Rβ expression in the sections of lungs from infected, diseased macaques (data not shown).
Upregulation of platelet-derived growth factor receptors in cultures of monocyte-derived macrophages
To understand whether SHIV infection could modulate PDGF-R expression, macaque MDM cultures were inoculated with the virus and examined at sequential intervals for modulation of PDGF-R using fluorescent-activated cell sorting and Western blots. Figure 4a shows that SHIV infection resulted in an increase in PDGF-Rα-positive cells from 17% in uninfected cells to 37% 5 days after infection, with a concomitant increase in the mean fluorescent intensity from 48 to 67. PDGF-Rβ-positive cells also increased by 53% by day 5, suggesting that infection in the macrophages caused an increase in both the number of receptor-positive cells and the number of receptors per cell. Western blot analyses for PDGF-Rβ in protein lysates isolated from infected MDM at days 3–6 following virus inoculation confirmed these findings. Figure 4b shows upregulated expression of PDGF-Rβ on infected MDM compared with uninfected cells. Similar upregulation of expression of PDGF-Rα was also noted (data not shown).
Since SHIV infection led to enhanced PDGF-Rβ expression on MDM, the functionality of these receptors was determined by analyzing the migration of SHIV-infected MDM towards a gradient of PDGF-BB. Chemotactic assays showed that PDGF-BB (at 0.1 ng/ml) had higher chemotactic activity for SHIV-infected MDM than for the uninfected MDM (Fig. 4c). At a higher concentration of PDGF-BB (1 ng/ml), there was statistically no difference in migration index between the infected and uninfected cells. This suggested that the infected cells were much more sensitive to the chemotactic signal than uninfected MDM.
We have previously demonstrated that development of pneumonia in SHIV-infected rhesus macaques was closely associated with increased levels of MCP-1 and CXCL10, and high viral loads in the lung parenchyma. In the present study, we examined the role of another well-recognized critical inflammatory factor, PDGF-B (a known inducer of the chemokine, MCP-1), and its receptors in SHIV-associated pneumonia. Overexpression of PDGF-B and its receptors has been shown to be linked to several inflammatory conditions, including fibrotic lung diseases, atherosclerosis, and gliomas [14–17,24–29]. Although PDGF has been implicated in HIV-associated pulmonary hypertension [30,31] and in AIDS-associated Kaposi's sarcoma [32,33], its role in SIV/SHIV-associated pneumopathy has never been investigated.
In the present study, lung tissues from 10 SHIV-infected rhesus macaques were evaluated for pathological changes and correlation of these lesions with PDGF-B/PDGF-R expression. Of the 10 infected macaques, six developed a spectrum of pathological changes including chronic interstitial pneumonia with multinucleated giant cells, granulomas, and, occasionally, fibrosis. Examination of lungs from these animals showed high levels of PDGF-B and its receptors, suggesting that this factor may have played an important role in the recruitment of inflammatory cells into the lung. We propose that multiple amplification steps, mediated by the PDGF/PDGF-R axis, were involved in the development of pneumonia and virus enhancement.
Macrophages were prominent among the inflammatory exudates in the affected lungs, and many of these cells were infected with the virus. To examine whether the infiltrating, virus-infected macrophages contributed to the increased pools of PDGF and its receptors, we analyzed the expression of the ligand and its receptors in these key cells in these tissues. Virus-infected macrophages in the lung tissue and in the bronchoalveolar lavage fluid of SHIV-infected macaques with pneumonia expressed both PDGF-B and its receptors. We had previously shown that SHIV infection of MDM resulted in increased PDGF-B expression . In the present study, we demonstrated that SHIV infection of these cells also upregulated the production of both PDGF-Rα and PDGF-Rβ. Thus, infection of macrophages with SHIV resulted in upregulation of both the receptors and their ligand, leading to an autocrine loop that could have amplified the pathological responses in the lung. Upregulation of the growth factor could, in part, be a possible explanation for enhanced virus replication in the SHIV-infected diseased lungs, since PDGF-BB is known to enhance SHIV replication . It is interesting to note that SHIV infection with P. carinii (macaques RMT5 and RHU5) correlated with decreased expression of PDGF-B compared with diseased animals within the same group. One possible explanation for this decrease could be decreased numbers of alveolar macrophages in these animals. This speculation stems from the earlier findings by Lasbury et al. [34–36] demonstrating that P. carinii infection in immunocompromised rats correlated with decreased macrophage burden in the lungs.
Since PDGF is a known chemoattractant for mesenchymal cells [37,38], we examined whether PDGF-B produced in the affected lungs could have recruited more virus-infected monocytes into the lung. Using MDM cultures, increased chemotaxis of SHIV-infected cells could be demonstrated to a gradient of PDGF-BB. This finding could have had implications in vivo, wherein increased numbers of infected monocytes could be recruited to a gradient of PDGF-BB expressed in the lung by the resident lung cells. Since SHIV infection is known to induce PDGF-B  and PDGF-Rβ expression, infected monocytes, once they entered the lungs, could self-perpetuate the amplification loop for recruitment of more monocytes into the lung. Another mechanism by which PDGF could have aided in recruitment of increased numbers of virus-infected inflammatory cells into the lungs, thereby contributing to lung pathology, stems from its function as an inducer of MCP-1 . Increased PDGF-B production in the lung could lead to increased MCP-1 levels, a known trigger for increased monocyte recruitment into the lungs. We have already demonstrated increased MCP-1 in the lungs of SHIV-infected macaques with pneumonia .
PDGF is known to play a role in the early stages of fibrogenesis in humans and rodents [14,16,40]. One interesting finding in this study was the development of mild fibrosis and collagen deposition in two of the six diseased animals. Interestingly in these two animals, fibrosis correlated with dramatic increase in PDGF-B expression in the lungs. Based on the fact that collagen deposition is known to occur late in the proliferative phase of the disease and that PDGF-B is mitogenic and also a chemoattractant for mesenchymal cells , we speculate that enhanced PDGF-B expression in the diseased animals may have contributed to the development of the fibrotic process by modulating the accumulation of mesenchymal cells in the alveolar walls. The PDGF/PDGF-R axis could, therefore, be an attractive target for therapeutic intervention aimed at abrogating virus replication in the lungs.
We thank Dr Sasahara, Mochida Company, Japan for providing us with the PDGF007 antibody.
Sponsorship: This work was supported by grants MH62969-01, RR-16443, MH-068212, MH072355, and DA020392-01 from the National Institutes of Health.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
PDGF; PDGF receptor; pneumonia; macrophages; SHIV