The cytokines and cytokine receptor examined were interleukin 2 (IL-2), IL-2-receptor (IL-2R), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), gamma interferon (IFN-γ) and tumor necrosis factor-α (TNF-α).
Heparinized whole blood was obtained from known HIV− and HIV+ individuals. All HIV+ subjects were homosexual men currently enrolled in the Multicenter AIDS Cohort Study (MACS) at UCLA (5
). The two HIV+ individuals used for detailed kinetic analyses had absolute CD4 T-cell counts of 168 and 312 cells/mm3
; the HIV+ group used for additional studies (n
= 8) had a mean absolute CD4 cell count of 376/mm3
, with a range from 213 to 476/mm3
. HIV− subjects were healthy heterosexual controls or HIV-seronegative participants in the MACS. PBMC were purified from whole blood samples by density gradient centrifugation over 60% Percoll gradients and cultured in complete RPMI 1640 medium (100 U of penicillin per ml, 100 μg of streptomycin per ml, 0.3 mg of glutamine per ml) with 10% human AB serum.
For kinetic studies, 106
PBMC in a total volume of 0.5 ml were placed in 12- by 75-mm tubes, allowed to rest without stimulation for 1 h at 37°C, and then stimulated with phytohemagglutinin (PHA) (Sigma) (5 μg/ml), or anti-CD3 monoclonal antibody (NEN Research Products) (200 ng/ml), as previously described (8
). PBMC pellets were collected at the indicated times (up to 72 h) poststimulation and stored at −70°C until RNA extraction. Culture supernatants (SN) were also collected at 24 and 72 h poststimulation and held at −20°C until assayed for cytokines or cytokine receptor.
RNA extraction and semiquantitative reverse transcription-PCR (RT-PCR) for cytokine gene expression were performed as previously described (7
), utilizing the primers shown in Table . Briefly, RNA was obtained by guanidinium isothiocyanate lysis and phenol-chloroform extraction, and 10 ng of total RNA was used for each RT-PCR. cDNA was synthesized using oligo(dT) priming and Moloney murine leukemia virus reverse transcriptase and then amplified using 0.2 μM 5′ and 3′ oligonucleotide cytokine or beta-actin primers, 2.5 μmol of AmpliTaq DNA polymerase (Perkin-Elmer Cetus), and a reaction mixture containing 10 mM Tris-HCl, 2.0 to 2.5 mM MgCl2
, 0.2 mM deoxynucleoside triphosphate (Perkin-Elmer Cetus), and 0.01 μM [α-32
P]dATP. Reactions were amplified by 25 cycles (beta-actin) or 35 cycles (cytokines) of denaturation at 95°C, with annealing and extension at 60°C. PCR products were identified by autoradiography following acrylamide gel electrophoresis, the radioactive bands were cut from the dried gel, and activity was determined by liquid scintillation counting. The activity obtained (counts per minute) for each cytokine reaction product was normalized to reflect total RNA content by using the activity obtained for beta-actin in the same sample.
TABLE 1 Primers for cytokine-specificRT-PCR
Concentrations of soluble IL-2R (Endogen), IL-4 (Genzyme), IL-10 (Immunotech), and TNF-α (Innogenetics) in cell culture SN were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol; IFN-γ (T Cell Diagnostics) was determined using a modified ELISA protocol (1
For eight HIV− and eight HIV+ subjects included in the kinetic studies described above, parallel experiments were performed on aliquots of PBMC from the same blood draw that were frozen in 10% serum–10% dimethyl sulfoxide according to MACS protocols (12
) and then thawed, stimulated, and cultured as described above. PBMC pellets and culture SN from frozen cells were collected at the same times, and tested in the same RT-PCR assays and cytokine ELISAs as the samples from fresh cells from the same subject.
The detailed kinetics of mRNA expression for each cytokine for each subject was represented by calculating the percentage of maximum mRNA expression obtained for a given cytokine at each time point over the 72-h time course. The mean percentage of maximum mRNA expression at each time point for each cytokine was determined for HIV− and HIV+ subjects and plotted as shown in Fig. and . Statistical analysis of the kinetic plots were performed by calculating the time axis centroid for each plot, which represented the mean time of mRNA expression. These mean times were then compared simultaneously for all six cytokines and cytokine receptor between HIV− and HIV+ subjects using a mixed-effects linear model (13
). Comparisons of mRNA values, mRNA ratios, and data for fresh versus frozen cells were performed using the nonparametric Wilcoxon rank sum test.
FIG. 1 Kinetics of cytokine mRNA expression following PHA stimulation of PBMC. Each cytokine mRNA value from a subject was represented as a percentage of the maximum mRNA obtained for that cytokine over that subject's time course. (A) Mean percent maximum mRNA (more ...)
FIG. 2 Kinetics of cytokine mRNA expression following stimulation of PBMC with anti-CD3 monoclonal antibody. (A) Mean percent maximum mRNA expression in the same HIV+ subjects as for Fig. A. (B) Mean percent maximum mRNA expression in (more ...)