 (TLR-4) complex (53, 54). CD14, the primary LPS receptor, exists in both cell surface membrane-bound and -soluble forms (27, 49). Membrane-bound CD14 (mCD14) is a 53-kDa GPI-linked cell surface antigen expressed on cells of the monocyte/macrophage lineage and to a lesser extent on neutrophils (58). Soluble CD14 (sCD14) likely represents cell surface CD14 that has been shed in response to either monocyte activation or differentiation (34). sCD14 can bind to circulating LPS-LBP complexes and can activate cells that do not normally express mCD14, such as CD14-negative monocytes and endothelial cells (24, 50).
CD14 expression is regulated by a number of factors, including the state of cell activation and the cytokines present in the microenvironment (8, 26, 30, 39, 52). Gamma interferon (IFN-γ) has been shown to induce CD14 expression in immature cell lines such as the myelomonocytic and monoblastic cell lines HL60 and U937 (20), although it has variable effects in more mature cell lines of the myelomonocytic lineage. Other cytokines that upregulate CD14 expression on monocytes include tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6, whereas IL-4 strongly reduces monocyte CD14 expression (48, 58).
The role of mCD14 and sCD14 in various disease states has been studied. Elevated levels of circulating sCD14 have been identified in patients with inflammatory conditions such as systemic lupus erythematosus (42), chronic active hepatitis (43), and septic shock (31). Immune suppression via administration of glucocorticoids has been shown to decrease mCD14 and sCD14 levels (41). In human immunodeficiency virus (HIV) infection, upregulated expression of mCD14, and increased levels of circulating sCD14 on monocytes (32, 40) and alveolar macrophages (56) have been reported. Elevated levels of sCD14 have been shown to correlate with disease progression in cases of HIV/AIDS (32, 40). Circulating LPS may have direct clinical significance in HIV disease, since LPS has been found to increase HIV production in latently infected cell lines via a CD14-dependent mechanism (4).
The molecular mechanism through which monocyte CD14 expression is upregulated in HIV infection remains unclear. Bacterial endotoxins (LPS), immunoregulatory cytokines (IFN-γ, TNF-α, IL-1, IL-4, IL-6, and IL-10) whose expression is upregulated in HIV infection (9, 10, 16, 18, 29, 36, 37), and circulating levels of HIV-regulatory antigens such as Tat, Nef, or gp120 may modulate CD14 expression on monocytes. HIV gp120 has been shown to modulate the expression of mCD14 expression on monocytes (57). In the present study, we investigated the effect of the HIV-regulatory proteins Tat and Nef, as well as immunoregulatory cytokines such as IL-10, on the expression of mCD14 on monocytic cells and the release of sCD14. The results suggest that mCD14 and sCD14 expression is enhanced in HIV-positive individuals and that this may be mediated in part through the effects of HIV-Nef and/or IL-10. We further investigated the mechanism underlying HIV-Nef-induced mCD14 expression on normal human monocytes. Our results suggest that HIV-Nef upregulates mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10, suggesting that LPS and HIV-Nef antigen may induce CD14 expression on monocytes by distinct mechanisms. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression in monocytes by interfering with IL-10-induced signals but not by blocking IL-10 production.
Reagents.HIV-Nef was purchased from two sources, namely, Research Diagnostics, Inc. (Flanders, N.J.), and ImmunoDiagnostics, Inc. (Bedford, Mass.). The HIV-Nef obtained from Research Diagnostics was a 140-kDa recombinant protein fused at the N terminus with β-galactosidase. The HIV-Nef obtained from ImmunoDiagnostics, Inc., was produced as a glutathione S-transferase fusion protein in Escherichia coli. The recombinant protein was purified by glutathione-affinity chromatography, and the purified fusion protein was cleaved by thrombin to produce a 27-kDa HIV-Nef protein. Endotoxin content was determined by BioWhittaker QCL-1000 Kit (Walkersville, Md.) essentially as described by the manufacturer. Both the HIV-Nef preparations were found to contain endotoxin. Endotoxin was removed by passing both HIV-Nef preparations through a column of polymyxin B agarose beads (Sigma) by using standard methods (38). The HIV-Nef preparations thus obtained contained <0.01 U of endotoxin per mg of protein. Endotoxin-free HIV-Tat was also purchased from ImmunoDiagnostics, Inc. Human recombinant IL-10 was purchased from R&D Systems (Minneapolis, Minn.). Neutralizing anti-IL-10 antibodies and isotype-matched control antibodies were purchased from Pharmingen (Mississauga, Ontario, Canada). Dexamethasone was purchased from Sabex (Boucherville, Quebec, Canada).
Isolation and culture of PBMC.Blood was obtained for isolation of peripheral blood mononuclear cells (PBMC) from healthy adult volunteers and HIV-positive individuals with CD4 counts ranging from 100 to 600 cells/μl after approval of the protocol by the Ethics Review Committee of the Ottawa General Hospital, University of Ottawa, Ottawa, Ontario, Canada. All patients were receiving reverse transcriptase inhibitors, and none had clinical evidence of acute infection at the time of specimen collection. PBMC were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Baie d'Urfe 130, Quebec, Canada) as previously described (14, 16). The cell layer, consisting mainly of mononuclear cells, was collected and washed three times in phosphate-buffered saline (PBS). PBMC were resuspended in Iscove modified Dulbecco medium (Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum (FBS; Gibco Laboratories, Grand Island, N.Y.), 100 U of penicillin/ml, 100 μg of gentamicin/ml, and 10 mM HEPES.
Isolation of monocytes from PBMC.Purified nonactivated monocytes were isolated by Optiprep density gradient medium (Nycomed Pharma AS, Oslo, Norway) (25). Briefly, Optiprep medium was mixed with blood and overlaid sequentially with lymphocyte specific density medium (1.078 g/ml), a solution with a density of 1.068 g/ml, followed by Hanks' balanced salt solution. The tubes were centrifuged at 600 × g for 25 min. The cell preparation obtained in the first fraction contained highly enriched (88 to 96%) CD14+ monocytes as determined by flow cytometric analysis. Contaminating T and B cells were eliminated by anti-CD2 and anti-CD19 antibody-conjugated immunomagnetic beads (Dynal AS, Oslo, Norway), respectively, as described earlier (16). These highly purified monocytes were not activated as determined by the comparable levels of HLA-DR expression on monocytes before and after isolation.
Cell stimulation and collection of culture supernatants.PBMC from HIV-infected and uninfected individuals (2 × 106 cells/ml) or monocytes from HIV-seronegative individuals (1.0 × 106 cells/ml) were stimulated with HIV-Nef (0.5 to 4 μg/ml), LPS (0.1 to 1 μg/ml), or IL-10 (1 ng/ml) in 24-well tissue culture plates (Falcon, Becton Dickinson, Lincoln Park, N.J.). Cells were harvested after 48 h and analyzed for CD14 expression by flow cytometry as described below. The supernatants were harvested after 48 h and frozen at −70°C. Supernatants were thawed at the time of analysis for measurement of soluble CD14 production by a commercially available enzyme-linked immunosorbent assay (ELISA) kit. The cell supernatants were also analyzed for IL-10 and IL-12p40 production by ELISA. All experiments were done at least three times and figures show representative experiments.
Measurement of sCD14 in the culture supernatants.Soluble CD14 was measured in the culture supernatant by ELISA by using a commercially available kit (IBL, Hamburg, Germany) as described by the manufacturer. Briefly, microtiter strips coated with oligoclonal antibody to human sCD14 were incubated with 50 μl of supernatant or 50 μl of the standards provided in the kit. The secondary biotinylated anti-CD14 antibody and the sample or supernatant were added simultaneously and incubated at room temperature for 2 h on an orbital shaker. The plates were washed three times after which peroxidase-labeled streptavidin was added for one h at room temperature on an orbital shaker. The plates were washed again three times. Soluble CD14 was detected with the tetramethyl benzidine (TMB) substrate solution. The color reaction was stopped with 1 M sulfuric acid, and the absorbance was read at 450 nm by using an ELISA reader (Bio-Rad Laboratories). The sensitivity of this assay was 2.2 ng/ml. Curve fitting and concentration calculations were performed by using the Microplate Manager 4.0 Software