Cytokines have a major role in the regulation and function of the immune system. T-helper (Th) cells produce several cytokines, and their subdivision is well documented (1-4). T-helper type-1 (Th1) cells produce interleukin 2 (IL-2), interferon γ (IFN-γ), and tumor necrosis factors α and β (TNF-α and -β), lymphokines which promote macrophage activation that results in delayed-type hypersensitivity. T-helper type-2 (Th2) cells produce IL-4, -5, -6, -9, -10, and -13 that help in the humoral immune response.
Cytokine regulation of the human immunodeficiency virus type 1 (HIV-1) infection may have great importance. In HIV infection, the detection of specific soluble antigens capable of stimulating proliferative T-cell response is complicated, even when the CD4 cell count is normal (5,6). Clerici and coworkers (7-9) have suggested that a switch from Th1 to Th2 cell response occurs in HIV-infected individuals progressing to AIDS. Their results show decreased levels of IL-2 and IFN-γ (Th1 cytokines) and increased levels of IL-4 and IL-10 (Th2 cytokines) production in recall antigen and mitogen-stimulated peripheral blood mononuclear cells (PBMCs) of HIV-infected individuals. Activation of Th2 cells could be responsible for the polyclonal activation of B cells as well as progressive immunosuppression in HIV infection (10).
Here, we present the results of mRNA expression of Th1 and Th2 cytokines in the CD8-depleted PBMCs of eight HIV-infected individuals, stimulated with leukoagglutinin (LA) and HIV-1 Tat and Rev peptides D26, D28, D57, and D60, previously found to induce a T-cell response in HIV-infected individuals with different human leukocyte antigen (HLA) class-II molecules (11,12). The method of reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect mRNA for IL-2, IFN-γ, IL-4, and IL-10. Our results show that there is no significant difference in the expression of any cytokine mRNA in the mitogen-stimulated cells of HIV-infected and noninfected individuals. However, for HIV-seropositive (HIV+) individuals, baseline expression of the above cytokine mRNAs was not detected, whereas it was detected in the cells of HIV-seronegative (HIV-) individuals. When the cells of HIV+ individuals were stimulated with the peptides, 70% of the cases showed IL-10 mRNA expression, 20% IFN-γ, and 10% IL-2, with no detection of IL-4 mRNA. Our results thus indicate that HIV-1 antigens Tat and Rev induce IL-10 mRNA expression in HIV-infected individuals.
The role of elevated IL-10 mRNA in HIV peptide-stimulated cells may inhibit cell-mediated immunity in HIV-infected individuals. Recently, IL-10 production has been shown to be increased in the PBMCs of HIV-infected individuals compared with noninfected individuals (13). IL-10 indirectly suppresses immune and inflammatory responses, and reduces antigen-specific proliferation and cytokine production of T cells (3,9,13), possible by downregulation of major histocompatibility complex (MHC) class-II expression on antigen-presenting cells (14,15). Therefore, increase in IL-10 may permit viral replication to go uncontrolled.
MATERIALS AND METHODS
Peripheral blood samples were collected from eight HIV-1-seropositive patients at Helsinki University Central Hospital, Department of Dermatological and Venereal Diseases (12). Clinical information including Centers for Disease Control and Prevention (CDC) stage, CD4 cell count, and in vitro T-cell response to a recall antigen, purified protein derivative of tuberculin (PPD), are shown in Table 1(11). Four HIV-1-seronegative healthy blood donors were used as negative controls.
Lymphocyte Isolation and Subtype Analysis
PBMCs were isolated from the heparinized venous blood of study individuals by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation and were cryopreserved. Samples of thawed (≈100% viability) PBMCs were depleted of CD8+ lymphocytes by incubating the cells for 10 min at room temperature with anti-CD8 magnetic immunobeads (Immunotech, Marseille Cedex, France) used according to the manufacturer's instructions for negative cell sorting. Cells binding to the beads were removed by 10-min contact with a magnetic concentrator (Dynal, Oslo, Norway). Cells remaining in the suspension, CD8- PBMCs, were used for stimulation and subsequent RNA extraction.
The phenotypic analysis of the sorted cells was performed on a FACScan (Becton Dickinson, Mountain View, CA, U.S.A.) flow cytometer. Cells were labeled with FITC-conjugated monoclonal antibodies Leu-3a + 3b (anti-CD4), Leu-2a (anti-CD8), Leu-16 (anti-CD20) (Becton Dickinson, San Jose, CA), and Tük-4 (anti-CD14) (Caltag, San Francisco, CA, U.S.A.).
To detect cytokine mRNA expression after stimulation with the specific HIV-1 antigens, 0.8-1.0 × 106 CD8- PBMCs were cultured in 1 ml RPMI-1640 medium (Gibco, Life Technologies, Paisley, UK) supplemented with 2 mM L-glutamine (Gibco) penicillin (100 U/ml; Gibco), streptomycin (100 μg/ml; Gibco), and 10% human AB+ HIV- serum in the absence or in the presence of two HIV-1 Tat synthetic peptides (D26 and D28, 50 μg/ml) and two Rev peptides (D57 and D60, 25 μg/ml), previously shown to be antigenic for T cells from HIV+ individuals (11,12). Immunogenic non-HIV peptide of rabies-virus glycoprotein was used at 25 μg/ml as a negative control peptide (16). After 48 h of incubation at 37°C, the cells were collected for mRNA purification.
To detect the cytokine mRNA expression by nonspecific activation, parallel cultures with LA (10 μg/ml; Pharmacia) were performed.
Highly purified intact mRNA was extracted from the above cultured cells by using the Dynabead mRNA Direct kit (Dynal) as instructed by the manufacturer. cDNA was made by incubation of RNA (denaturated at 65°C for 10 min) with 0.2 μg random oligonucleotide hexamers (Pharmacia Biotech, Uppsala), 1 U RNAguard RNAse inhibitor (Pharmacia), 1.8 mM dNTP, 200 U Moloney murine leukemia virus RT (Pharmacia), 80 μg/ml bovine serum albumin and RT buffer (68 mM KCl, 9 mM MgCl2, 15 mM dithiothreitol, and 45 mM Tris-HCl, pH 8.3) for 60 min at 37°C in a final volume of 15 μl. The solution was heated to 90°C for 5 min, cooled to 5°C, and stored at -70°C until used in PCRs.
PCR amplification was performed using oligonucleotide primers specific for IL-2, IFN-γ, IL-4, IL-10, and β-actin (Table 2). All primers were synthesized from the sequence data published previously (17-21). Primers for ubiquitously expressed β-actin gene were used as a control for the efficiency of the PCR. Primer specifity was determined by the production of a PCR product of the predicted size (Table 2). PCR was performed using aliquots of the synthesized cDNA to which was added 200 μM dNTP (Boehringer Mannheim, Mannheim, Germany), 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1% Triton X, 2 U Taq DNA polymerase (Boehringer Mannheim), and 0.4 μM of each primer in a total volume of 25 μl. The reaction mixture was overlaid with a drop of mineral oil, and amplification was performed for 40 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min. Positive control for cytokine genes expression included LA-stimulated HIV- lymphocyte cDNA. Amplification of deionized water was used as a negative control in each PCR to ensure that reagents were not contaiminated. Following amplification, 7 μl of the hybridized product was separated at 80 V for 2 h in 1.8% agarose in 1× Tris-acetate buffer containing 0.5 μg/ml ethidium bromide. A Hinf I digest of pBR322 DNA size marker was run in parallel to provide molecular weight markers. The gels were inspected and photographed under ultraviolet transilluminator.
Of the eight HIV-1-infected individuals tested in these studies, five had a CD4 cell count of >500/mm3, whereas three had a count of <500/mm3(Table 1). The clinical status was as follows: one patient had CDC stage-II disease, six had CDC stage-III disease, and one had CDC stage-IVD disease. All HIV+ individuals had been vaccinated in childhood with bacille Calmette-Guérin, and six of the eight individuals had retained their in vitro T-cell proliferative response to a recall antigen PPD (Table 1), as defined in our previous studies (11).
Tat and Rev synthetic peptides used for the stimulation of our cell cultures were previously shown to induce a T-cell response in HIV-infected individuals with different HLA class-II molecules (11,12). Two peptides of HIV-1 Tat, D26 [amino acids (aa) 17-32] and D28 (aa 33-48) induced a proliferative T-cell response in 57% and 38% of individuals tested. HIV-1 Rev peptides D57 (aa 9-23) and D60 (aa 33-48) stimulated a proliferation of T cells in 50% and 44% of HIV+ individuals. These responses were HIV-1 specific, as no positive response was seen in HIV- control persons. A non-HIV control peptide was derived from rabies-virus glycoprotein sequence (aa 32-44), previously shown to contain a T-cell antigenic site (16).
As has been shown in earlier studies (22,23), CD8+ T cells can produce the same pattern of cytokines as do Th1 and Th2 cells; CD8+ cells were eliminated from our PBMC cultures. PBMCs were depleted of CD8 cells by anti-CD8 magnetic immunobeads, and the subtype analysis of the remaining PBMCs showed that they contained ≈80% CD4+ T cells, 10% B cells, and <5% monocytes, with no detectable CD8+ cells.
Cytokine Profiles of Mitogen-Stimulated and Unstimulated CD8- PBMCs
Figure 1a shows the pattern of cytokine gene expression upon stimulation with LA in eight samples of CD8- PBMCs from HIV+ individuals (letter codes) and four samples of HIV- individuals (numbers). Amplification with β-actin primers produced uniform strong signals for all samples, indicating consistency in RNA extraction, cDNA synthesis, and PCR efficiency in the different samples. PCR products specific for IL-2, IFN-γ, IL-4, and IL-10 were observed in almost all HIV+ samples and all HIV- samples polyclonally activated with LA. The mRNA for IL-2 and IL-4 was detectable in 88% (seven), mRNA for IFN-γ in 63% (five), and for IL-10 in 100% of eight HIV+ individuals tested.
Baseline cytokine expression was analyzed in unstimulated CD8- PBMCs of six HIV+ individuals and compared with that observed in HIV- control individuals. As Fig. 1b shows, the cells from four HIV- individuals spontaneously expressed mRNA for IL-2 and IL-10. IL-4 mRNA was detected in one individual, while IFN-γ was absent from all. Except for one HIV+ individual having a signal for IL-10 mRNA, no cytokine mRNA was detected in any other HIV+ individual, although β-actin-specific PCR products were obtained for each patient sample, and positive control reactions produced a consistent yield of each cytokine-specific product (Fig. 1b).
Cytokine Profiles of HIV-1 Tat and Rev Peptide-Stimulated CD8- PBMCs
Figure 2 shows the β-actin and cytokine gene expression induced by HIV-1 Tat and Rev peptides D26, D28, D57, and D60. The β-actin gene was used to asses the integrity of the RNA and was detected in all of the samples. Positive control samples (lane +) gave detectable signals for all cytokines, while negative control samples (lane -) remained without any signal, confirming that the PCRs were successful. The IL-10 gene expression was induced with each peptide. IFN-γ mRNA was also detected in some samples stimulated with all four peptides, whereas only Rev peptides D57 and D60 (Fig. 2c and d) induced IL-2 mRNA expression. Interestingly, IL-4 mRNA expression was not present in any sample, although an IL-4-specific amplification band was obtained from the positive control samples (Fig. 2, lane +). Neither of the two negative control peptide-stimulated cell cultures showed any cytokine mRNA expression (data not shown). The peptide-stimulated cells of HIV- control individuals showed the same pattern of cytokine gene expression as seen for the baseline production of the unstimulated cells (data not shown).
Table 3 shows the frequency of the cytokine mRNAs expression induced by the Tat and Rev peptide stimulation of CD8- PBMCs of HIV-seropositive individuals. IL-2 mRNA was detected in two (10%), IFN-γ in four (20%), IL-4 in none of 20, and IL-10 in 14 (70%) of the 20 samples tested. There was no correlation between any cytokine mRNA expression and the T-cell response to recall antigen PPD, the CD4 cell number, or the CDC stage of the patients (Table 1). Regardless of the clinical data, all patients except D, whose cultures did not produce any cytokine gene signal, had the IL-10 mRNA signal, and none showed the IL-4 mRNA signal.
In HIV infection, where the immune system gradually deteriorates, cytokines may play an important regulatory role. Data on this issue are, however, very contradictory. First, many groups have reported a decline in Th1-cell-type cytokine (IL-2 and IFN-γ) production in HIV-infected individuals progressing to AIDS (24-26). Further, Clerici and Shearer (7) have suggested a switch from Th1 to Th2 cell response, where increased levels of IL-4 and IL-10 follow loss in IL-2 and IFN-γ production in response to recall antigens, or mitogen, in HIV-infected individuals progressing toward AIDS. Also, other investigators have reported increased IL-10 production in HIV+ individuals compared with HIV- individuals (13,14). Maggi et al. (4) have presented data where a shift from Th1 to Th0 cell response occurs in HIV infection, and the cells produce both Th1- and Th2-type lymphokines. The data are thus numerous and, although quite different, all investigators agree about the importance of cytokine-mediated regulation of HIV infection.
This study investigated the cytokine gene expression in CD8- PBMCs of HIV-infected individuals stimulated with mitogen LA and with HIV-1 Tat and Rev synthetic peptides. RT-PCR was used to detect Th1-cell-type cytokines IL-2 and IFN-γ and Th2-cell-type cytokines IL-4 and IL-10. The cytokine mRNA expression profiles of HIV+ individuals were different in CD8- PBMCs stimulated with Tat and Rev peptides D26, D28, D57, and D60, compared with polyclonally stimulated or unstimulated cells. The data presented here are qualitative, although β-actin mRNA signals were uniform for all samples tested by RT-PCR. Upon polyclonal stimulation with LA, the cells of HIV+ persons produced Th1- and Th2-cell-type cytokine mRNAs comparable to the control group (HIV-). Therefore, our data disagree with the previous reports on the disability of IL-2, IFN-γ, and IL-4 production by mitogen-stimulated cells in all, or in deteriorated, stages of HIV infection (4,7). Baseline expression of cytokine genes was not detected in HIV-infected persons, except in one individual having a signal for IL-10 mRNA and in HIV- control individuals showing strong signals for IL-2 and IL-10 mRNAs. This result indicates that some level of dysregulation in spontaneous cytokine production exists in HIV+ compared with HIV- persons. Finally, IL-10 mRNA expression was upregulated in HIV+ individuals whose cells were stimulated with all HIV peptides tested. The peptides used for the stimulation were previously shown to be antigenic for T cells in up to 60% of HIV+ individuals with different DR molecules and very well conserved between different HIV isolates (11,12). The repeated amplification demonstrated clearly the presence or absence of cytokine gene expression in the stimulated cells, compared with unstimulated cells. The failure of the immunogenic rabies peptide to induce cytokine mRNA expression demonstrates that cytokine gene expression was HIV peptide specific. Of all HIV peptide-stimulated cultures, 70% showed IL-10 mRNA expression, 20% IFN-γ, and 10% IL-2, with no detection of IL-4 mRNA. Reduced production of IL-2 and IFN-γ has been observed by others (24-26), especially in the T cells of AIDS or AIDS-related-complex patients. Also, IL-10 production in the later stages of HIV infection, when IL-2 and IL-4 have declined, is suggested (7). No correlation between IL-10 production and the clinical stage of the patients, the CD4 cell number, and the ability to respond to a recall antigen PPD was observed.
Although IL-4 mRNA was not detected in any of the peptide-stimulated samples, the high frequency of IL-10 mRNA (70%) and the low frequencies of IL-2 and IFN-γ mRNAs (10% and 20%) indicate a Th2 pattern of cytokine gene expression. Indeed, other reports have indicated that IL-4 mRNA detection is very difficult (21,27) and that recall antigens do not stimulate detectable IL-4 production (7). To eliminate the possibility of CD8+-cell-derived production of Th1- or Th2-type cytokines (22,23), CD8 cells were depleted. Therefore, we assume that IL-10 gene expression in HIV peptide-stimulated cultures occurs in Th2-type cells, as others (7-9,14,27) have shown that IL-10 is a Th2-response cytokine.
Although the IL-10 protein level was not measured in these studies, the elevated IL-10 mRNA in HIV peptide-stimulated cells indicates that this interleukin would be transcribed in the cells. If that is so, IL-10 induced by HIV antigens Tat and Rev may be responsible for the inhibition of the cell-mediated immunity (for example, cytotoxic T lymphocytes) often seen in HIV-infected individuals. It was shown that IL-10 addition to Th1 and Th2 clones reduces proliferation to both specific antigen and mitogen, and to antigen-induced production of IFN-γ, IL-4, and IL-5, by downregulating class-II MHC expression on antigen-presenting cells (15). Clerici et al. (9) already showed that a low or lacking proliferative T-cell response to HIV-1 Env peptides could be restored with anti-IL-10, but not with anti-IL-4 antibodies. In HIV infection, recognition of processed HIV peptides by helper T cells will possibly lead to the production of IL-10 instead of IL-2 and IFN-γ, and this may lead to the T-cell anergy and B-cell hyperactivation often seen in HIV infection.
Acknowledgment: This work was supported by the Academy of Finland. The technical assistance of Ms. Tuula Myllymäki is gratefully acknowledged. We thank Ms. Auni Collings for editing the text.
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