Adaptive immunity to rhinoviruses: sex and age matter

We recruited healthy adult volunteers with no history of asthma or other lung diseases. Females (n = 32) and males (n = 31) were divided into those aged up to 50 years, and those aged 52 years and older, as the median age of menopause in Australia is 51 yrs [22]. The women ≥52 years were not undertaking any hormonal therapy. One woman in the ≤50 year old group was currently taking the oral contraceptive pill. The subjects of the study had skin prick testing (SPT) to a panel of common allergens and were questioned about symptoms of allergy and lung diseases. The study was approved by the Princess Alexandra Hospital and the University of Queensland Human Research Ethics Committees, and informed consent was obtained from each subject.

Ohio HeLa cells and the major group RV strain RV16 were kindly donated by Professor Phil Bardin, Monash Medical Research Centre, Melbourne, Australia. RV stocks were generated by passage in Ohio HeLa cells as described previously [23] followed by purification over a sucrose gradient [24]. To determine the optimal concentration of RV, Ohio HeLa cells were seeded into a 96 well plate at a density of 1 × 10⁴ cells per well in 150 μl RPMI containing 2% low endotoxin fetal bovine serum (FBS; Bovogen Biologicals, Victoria) and allowed to adhere overnight. The following day 10-fold serial dilutions of virus was added in triplicate to the cells at 50 ul/well and cultured at 37°C for 6 days. The media was removed and cells stained with 0.1% crystal violet solution in PBS to determine the amount of virus required to infect 50% of cells (TCID₅₀).

PBMC were isolated from heparinised blood by density gradient centrifugation as previously described [11], and cultured at 1 × 10⁶ PBMC in 24 well culture plates at a final concentration of 2 × 10⁶ cells per/ml together with RV16 at a multiplicity of infection (MOI) of one; i.e. one virion per cell. Control cultures contained medium alone. RPMI media was supplemented with antibiotics, 2-mercaptoethanol and either 10% foetal calf serum (FCS; innate immune studies) or 5% autologous plasma (adaptive immune studies). Extensive comparative experiments showed that both FCS and autologous plasma supplemented media induced identical innate immune responses, though autologous plasma was preferred for adaptive immune studies in order to minimise foreign antigen exposure and because autologous plasma supplemented media was associated with consistently higher adaptive IFNγ synthesis (data not shown). The experiments that required depletion of antigen experienced/memory T cells from PBMC were performed using CD45R0+ immuno-magnetic beads (Miltenyi Biotech), according to the manufacturer's directions. Cultures were incubated at 37°C with 5% CO₂ and supernatant was harvested for cytokine quantification by ELISA. Cell pellets were stored in 'RNA-protect' (Qiagen) until RNA was extracted. Initial time course experiments indicated that optimal expression of mRNA for IFN-stimulated genes and Th1-polarising genes was at 6 hours post-stimulation. The optimal time point for IFN-gamma-inducible protein 10 (IP-10, also known as CXCL10) secretion was at 24 hours post-stimulation while the optimal time point for the adaptive cytokines IFNγ and IL-13 was at 5 days post-stimulation.

IP-10, IFNγ and IL-13 ELISAs were performed using commercially available paired antibodies and recombinant cytokines (Becton Dickenson, Franklin Lakes, NJ). The limit of detection was 15.6 pg/ml for IP-10 and IFNγ, and 7.8 pg/ml for IL-13. IFNα was assayed via ELISA kit (PBL Interferon Source, Piscataway, NJ) according to the manufacturer's instructions; the limit of detection for IFNα was 9.7 pg/ml.

RNA was extracted using RNeasy Spin kit and RNA was reverse transcribed using Quantitect reverse transcription kit (Qiagen, Hilden, Germany), according to manufacturer's instructions. Gene expression was investigated by quantitative PCR (qPCR), using the ABI 7900 HT (Applied Biosystems, Foster City, CA USA) with Quantitect SYBR green (Qiagen). As the expression levels in unstimulated cultures were negligible and gene expression was induced by RV16, quantitation was achieved using standard curve analysis [25]. Ten-fold serial dilutions of PCR product standard were used to create standard curves for the gene of interest and the reference gene UBE2D2. UBE2D2 was initially identified as a stable reference gene in CD4+ cells [26] and subsequently assessed in-house to be stably expressed in total PBMC in the absence and presence of rhinovirus 16, using the method described by Silver et al [27]. The resultant ratio of gene of interest to reference gene were natural log transformed to normalise the data, allowing parametric statistical analysis to be performed. Table 1 shows the primers used to amplify cDNA. Copy numbers were determined by 10-fold serial dilution of PCR product standard and normalized to the reference gene UBE2D2 [26]. Data are expressed as a ratio of stimulated to control (unstimulated) samples.

Statistical analysis was performed using SPSS 18 (IBM SPSS Inc., Chicago, IL, USA). After the data was log transformed, it was found to be normally distributed. Group comparisons were therefore made using paired two-tailed t-test, with p < 0.05 considered significant. Correlations between variables were made using Pearson's correlation. Raw data is presented as medians and interquartile range, while transformed data is presented as mean ± standard error of the mean (SEM).