Introduction
Chemokine receptors have a pivotal role in HIV-1 cell entry [1-4], and considerable focus has been placed on the role of this interaction during transmission [5-7]. The proposed key site of heterosexual acquisition of HIV-1 in females is via the cervix [8], and either the inner surface of the foreskin or the urethra in males [9]. Data from previous studies of chemokine receptor expression in the female genital tract are ambiguous. One report, using immunocytochemical staining, revealed the presence of high levels of CXCR4 and CCR3 in the vagina and cervix of both macaques and humans [10]. Later work revealed higher levels of CCR5 expression compared to the other chemokine receptors [6,9], and this has given rise to the current dogma that differential transmission of CCR5-tropic (R5) isolates is due to higher expression of CCR5 within the genital tract epithelia. These latter studies focussed on expression in either cervical biopsies from female participants or circumcision operation-derived foreskin material in males. Consequently these data might not accurately reflect expression levels at the surface of the mucosal epithelium.
The ligands for these receptors [RANTES (regulated upon activation normal T cell expressed), MIP-1α (macrophage inflammatory protein) and MIP-1β for CCR5 and SDF-1 (stromal cell-derived factor) for CXCR4] have been shown to suppress HIV-1 infection both in vitro [5,11] and in vivo [12]. Therefore, expression of these might also play a role in transmission. Whilst β-chemokine secretion (i.e. CCR5 ligands) has been investigated in the cervico-vaginal fluids of HIV positive women [13], there is a paucity of data available on chemokine mRNA expression within the male urethra and in the female genital tract epithelia.
Competitive reverse transcription (RT)-PCR enables precise quantification of mRNA in small quantities of cells [14,15]. We have developed a method for the accurate quantification of various mRNA transcripts present in samples collected from the epithelia of the male urethra and female cervix. The method has been applied to a cohort of sexually active men and women attending an urban genitourinary medicine (GUM) clinic in the UK. Levels of the HIV coreceptors, chemokines and cell type-defining markers in the male urethra and female endo- and ectocervix have been measured and compared.
Methods
Patient recruitment and sample collection
Male and female patients attending the Nottingham City Hospital GUM clinic were invited to join the study and consent appropriate to local regulations was ascertained. Male enrolees granted permission to analyse the urethral swab (cotton tipped with plasticized paper shaft; Technical Service Consultants Ltd, Heywood, UK) taken as part of their routine examination, while females were asked to undertake an additional cervical scrape (Cervex-brush cervical cell sampler, Rover Medical Devices B.V., Oss, The Netherlands). Patients were examined for genital ulcers, warts, balanitis and epididymitis, vulvo-vaginitis and cervicitis, and routine tested for non-gonococcal urethritis, Chlamydia trachomatis, Neisseria gonorrhoeae, trichomonal vaginosis, Candida spp., bacterial vaginosis, herpes simplex virus, syphilis and HIV, as appropriate. Sample collection implements were immediately placed in 1-3 ml RNA lysis buffer (100 mM Tris-HCl pH 7.6, 500 mM LiCl, 10 mM EDTA pH 8.0, 15 mM LiDS; 5 mM dithiothreitol) and resulting lysates stored at -80°C. Peripheral blood mononuclear cells or liver tissue were used as a source of mRNA for construction of the PCR competitors.
mRNA extraction and cDNA synthesis
Between 150 and 1000 μl frozen cell lysates were thawed, diluted with fresh lysis buffer to yield a final volume of 1 ml, then mRNA extracted using 20 μl magnetic beads from the Dynabeads mRNA DIRECT Micro kit (Dynal A.S. Oslo, Norway), as described by the manufacturer. Purified bead-bound mRNA was resuspended in 25 μl RT buffer (50 mM Tris-HCl 75 mM KCl, 3 mM MgCl2, 50 mM DTT; pH 8.3 at 25°C; Promega, Southampton, UK) containing 15 units DNaseI (Roche, Lewes, UK) and incubated at 37°C for 5 min. RNA preparations were heated to 70°C for 5 min then snap-cooled on ice. Between 5 and 20 μl mRNA was used as a template in a 50 μl RT reaction containing 400 U Moloney murine leukaemia virus reverse transcriptase (Promega), 20 U RNase inhibitor (Ambion, Huntingdon, UK), 10 nM each dNTP (Roche) in 1 × RT bffer (Promega). cDNA synthesis was performed at 42°C for 1 h in a thermal cycler and stored at 4°C as recommended by the manufacturer.
Primer design and synthesis
Primers (Table 1) were designed using the Primer3 program [16] and the Molecular Biology Workbench, version 3.2 (http://workbench.sdsc.edu/). Control hypoxanthine phosphoribosyl transferase (HPRT) sense and antisense primer sequences used were those described previously [15]. Sense and antisense primers were additionally synthesised with a 5′ EcoRI restriction site (GGAATTC) and 5′ BamHI restriction site (additional GCGGATCC) to facilitate directional cloning. A further variation of each sense primer was synthesised to contain a 5′ fluorescent label in one of three colours to allow co-electrophoresis and detection, as follows: FAM [Bonzo, CD14, CD19, MIP-1α, RANTES and T-cell receptor (TCR)α], HEX (CCR3, CCR5, CD4, MIP-1β and SDF-1), TET (CXCR4 and HPRT).
Competitor construction and quantification
Deletion mutant competitive PCR products were synthesised by re-amplifying coreceptor or chemokine-specific PCR products with the appropriate competitor primers (Table 1). The competitor primers were designed to introduce a 4-5 bp deletion into the amplimer. Deletion products were cloned into the pTRI-amp-18 vector (Ambion).
Purified competitor plasmid stocks were quantified initially by fluorimetry and then more accurately by limiting twofold dilution end-point PCR [17-19], in a 55-cycle single-round PCR amplification (see below). Competitor plasmids were then readjusted to various working concentrations (in 10 ng/μl polyuridylic acid single stranded RNA carrier) as determined by the end-point dilution quantification.
Competitive PCR
One microlitre cDNA was used as a template in a 25 μl PCR reaction containing 50-500 copies of the appropriate plasmid competitor, 0.6125 U Hot Star Taq (Qiagen, Crawley, UK), 100 μM dNTPs (Roche), 200 nM forward and reverse primer diluted in 1 × PCR buffer (Qiagen). PCR was carried out as follows: 95°C for 15 min, then 55 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 90 s, with a final extension step of 72°C for 10°min after which products were held at 4°C.
Between 1 and 4 μl PCR amplification products (empirically determined by preliminary electrophoresis and product peak height evaluation) specific to a clinical sample were combined, precipitated, washed and resuspended in 50 μl water. Products were electrophoresed as previously described [15], then quantified and normalized by HPRT content by the following calculation: unknown concentration = [(endogenous peak area/competitor peak area) × competitor copy number]/HPRT copy number/μl cDNA. Where possible, analyses were repeated if the largest peak of a sample fell below 1000 fluorescence units in height. However not all specimens could be tested for every gene due to limited RNA availability. The lower thresholds of detection for chemokine receptor, ligand and cell-type marker transcripts were 0.29, 6.73 and 0.63 copies per HPRT transcript, respectively. Statistical analysis was performed using the Graphpad Prism and Microsoft Excel software packages.
Fluorescence activated cell sorting (FACS)
Female genital tract cytobrush specimens were immersed in 25 ml RPMI media, supplemented with 5% foetal calf serum and 0.1 % sodium azide then prepared for FACS analysis as published previously [20]. The following fluorochrome-labelled antibodies were used for staining: CD45 (pan-leukocyte marker, phycoerythrin-cyanin5 labelled, Beckman Coulter, High Wycombe, UK), CCR5 (phycoerythrin labelled, R&D Systems, Abingdon, UK) and CXCR4 (fluorescein labelled, R&D Systems). Median CCR5 and CXCR4 fluorescence was corrected for autofluorescence by subtraction of median fluorescence generated by an isotype control. Statistical analysis was performed using the Graphpad Prism software; samples yielding negative values when corrected for autofluorescence were assigned an arbitrary cut-off value of 0.1.
Results
Patients
Of 16 male patients (age range, 19-74 years), 11 had evidence of one or more infections, whilst five were negative for all infections. Of the 24 female patients (age range, 17-55 years), 13 had genital tract infections, whilst 11 were negative for all conditions assessed (clinical information available from the authors upon request).
Competitive PCR linearity and reproducibility
To determine the dynamic range and linearity of the quantitative RT-PCR, replicate HPRT amplifications were performed using 125 copies of the HPRT competitor plasmid and a 6-log range of cDNA dilutions as target DNA. A linear relationship of target to competitor ratio was seen within an approximately 3-log range of cDNA dilutions. Similar trends were observed for all pairs of competitor and targets amplified (not shown). Assay reproducibility was confirmed by repeating quantitative competitive amplification of seven samples and 13 types of competitive reaction. The mean variation was less than 0.42 logs with a standard error of 0.39.
HIV coreceptor, chemokine ligand and cell-type marker transcription in the genital tract
Expression of the HIV coreceptors CCR5, CXCR4, CCR3 and Bonzo, the CCR5 ligands RANTES, MIP-1α, MIP-1β and CXCR4 ligand SDF-1 and the cell-type markers CD4, CD14, TCRα and CD19 in the male urethra and female ecto- and endocervix was investigated (Fig. 1). CXCR4 was the predominant chemokine receptor expressed in the male urethra and female endo- and ectocervix (Fig. 1a and d). Median copy numbers were significantly higher than median values for CCR3 (P = 0.002, Wilcoxon matched pairs analysis) and Bonzo (P = 0.0391) in males, and CCR5 (P = 0.0039), CCR3 (P = 0.002) and Bonzo (P = 0.0059) in females. The descending order of median value of chemokine receptors (CXCR4, CCR3, Bonzo and CCR5) was identical at both the male and female genital tract sites sampled. CCR3 and Bonzo were also expressed at a significantly higher level than CCR5 in females (P = 0.0078 and 0.0322, respectively). The majority of the CCR5 values were undetectable or near the cut-off level. By contrast, CXCR4 was detected in all but one of 15 (7%) male and two of 22 (9%) female patients over a considerable range (> 2 logs in both sexes). CCR3 was also commonly detected with only three male (25%) and female (17%) patients' values falling below the cut-off.
A similar analysis of chemokine mRNA expression in the same samples showed considerable differences in the level of RANTES, MIP-1α and MIP-1β expression (Fig. 1b and e). In contrast SDF-1 mRNA was not detected in either male or female genital tracts. Samples frequently lacked detectable inflammatory chemokine MIP-1α and MIP-1β mRNA expression. By contrast RANTES mRNA was commonly detected in samples from both sexes, with only one of 16 (6%) male and three of 21 (16%) female samples having undetectable RANTES mRNA expression. That said, MIP-1α and MIP-1β showed approximately 1 log higher maximal levels of expression in the male urethra than RANTES, but all three displayed similar ranges in the endo- and ectocervix in females. The only notable statistical difference in CCR5 ligand medians was between MIP-1α and MIP-1β in females, where MIP-1α was generally seen to predominate (P = 0.0017).
To determine the cellular composition of the epithelial samples as well as to identify the likely sources for coreceptor and chemokine ligand expression, mRNAs corresponding to various cell-surface molecules were measured. Detection rates and expression ranges were similar in both male and female samples. Similar ranges of values and ranking was seen in both the male and female genital tracts (Fig. 1c and f). The CD14 monocyte-macrophage marker was detected in 13 of 14 (93%) males and in 19 of 21 (90%) females. CD4 expression was detected in 11 of 15 males (73%) and 11 of 17 (65%) females, whereas the pan T-cell marker, TCRα, was detected in 13 of 14 (93%) males and 17 of 24 (71%) females. In contrast the B-cell marker CD19, an indicator of blood contamination [20], was rarely detected in either male or female samples. The median level of CD14 expression was significantly higher than CD4 expression for both male CD14 (P = 0.0023) and female samples (P = 0.0046). Cell surface receptor expression was not associated with HPRT expression, an indicator of overall mRNA quantity (data not shown).
Correlation of HIV coreceptor and endogenous ligand transcription within the genital tract
Non-parametric Spearman's rank correlation analysis showed that CXCR4 expression was correlated to CCR3 (P = 0.0024), RANTES (P = 0.0057) and MIP-1β (P = 0.0072) expression in the male urethra. In the endo- and ectocervix, CXCR4 expression was also significantly associated with that of MIP-1α (P = 0.0004), MIP-1β (P < 0.0001) and to a lesser extent CCR3 (P = 0.0491). CCR3 was correlated with RANTES (P = 0.0269) and MIP-1α (P = 0.01) expression in males alone, whilst Bonzo correlated with RANTES (P = 0.0074) and MIP-1β (P = 0.0447) in the endo- and ectocervix only. Correlation analyses for CCR5 expression were not performed due to the high number of censored data points. Strong correlation was observed between MIP-1α and -1β mRNA expression in the male urethra (P = 0.0032) and female endo- and ectocervix (P = 0.0007), and between RANTES and MIP-1β (P = 0.0021) in men.
Correlation of HIV coreceptor and target cell marker transcription within the genital tract
Spearman's rank correlation analyses (Table 2) showed that CXCR4 expression was correlated with CD14 expression in both males and females (P < 0.0001, Fig. 2), as was CCR3 (male, P = 0.0001; female, P = 0.0293). CXCR4 expression was also correlated with CD4 expression in men (P = 0.0235) and women (P = 0.0414), but not with the other immune cell markers investigated. Again, correlations with CCR5 were not performed due to the low number of data points that were above the detection limit.
Analysis of cell-surface expressed CCR5 and CXCR4 in the female genital tract
To verify the finding that CXCR4 expression was significantly higher than CCR5, samples obtained from the female genital tract were analysed by FACS (Fig. 3). CXCR4 expression on both total and CD45-positive cells was significantly higher than CCR5 expression, which is in good agreement with the data obtained for mRNA transcription analysis. Low cell numbers obtained in some samples precluded comparative analyses of surface and mRNA expression in the same samples and a similar analysis on male samples.
Discussion
CCR5 and CXCR4 are the principal coreceptors for cellular infection by HIV [21], and their expression at genital mucosa must play a key role in establishing infection post-inoculation. It has been proposed that the predominantly R5 HIV infection following transmission [22,23] may arise through predominant expression of CCR5 [6,9]. However, we have shown that expression of CXCR4, CCR3 and Bonzo mRNAs in samples obtained from genital epithelia are significantly higher than that observed for CCR5. Importantly, FACS analysis of samples obtained from the female genital tract confirmed a higher level of expression of CXCR4, and good correlation between mRNA and cell-surface receptor expression has been reported previously [6,24]. We have also presented the first analysis of coreceptor expression in the male urethra. These data indicate that the distal tip of the male urethra is an anatomical site potentially highly susceptible to HIV infection.
We did not control for menstrual cycle status or contraceptive use as previous work has shown that genital tract intraepithelial immune cell density and distribution is not affected significantly by either [25-28]. Also, as recruitment was from a GUM clinic, we consider it was representative of a range of individuals potentially at risk for HIV infection. Epithelial samples from the male and females subjects were obtained using methods previously shown to be effective means of sampling epithelial and intra-epithelial cells [20,29-34]. We sampled the epithelia from both the endo- and ectocervix as both harbour HIV-permissive cells [7,8,35,36]. Although the anatomy of these regions differs markedly (reviewed in [37]), both could be exposed to HIV-1 infection. Detection of the B-cell marker CD19 in only four samples is congruent with previous data [8,20,25,38] and confirms that our samples had little, if any, blood contamination. Notably three of these four positive samples were negative for CCR5.
A potential pitfall of our approach was that we were not able to associate coreceptor expression with cells permissive for HIV infection, and this will require a more in-depth analysis of cell subsets. That said, previous studies have shown that coreceptor expression in the genital tract is restricted to target cells for HIV infection [9,10,29,39,40]. We observed strong correlations between CXCR4 and CCR3 with the monocyte-macrophage marker CD14 in both males and females.
Our finding that CCR5 mRNA expression is lower than both CXCR4 and CCR3 within the female genital tract is at odds with that previously published by Patterson and colleagues [6,9]. We have verified that these differences are not due to discrepant amplification efficiencies of the primers used by the two groups (not shown). One important reason for the observed difference could be that Patterson's group detected mRNA expression in homogenized punch biopsies, which will represent expression profiles in cells present in the lamina propria and the submucosa as well as the epithelia [41]. We focussed on samples obtained from the epithelia only, as we wished to determine coreceptor repertoire in the cells exposed to HIV in an intact mucosal epithelium.
Importantly our data is in line with an immunohistochemical study of coreceptor expression in the genital tracts of macaques and humans [10]. This study reported detection of a large number of CCR3 and CXCR4 expressing cells (which were most likely intraepithelial macrophages or Langerhans' cells) within the cervical mucosa of both uninfected macaques and humans, whereas CCR5-positive cells were rare. This contrasted with both rectal mucosa and lymphoid tissue where significant numbers of CCR5-expressing cells were detected [10].
Selective transmission of R5 isolates caused by selective CCR5 expression in the genital tract has become widely accepted dogma [6,9,22,42]. However, rather than selective transmission being imposed at the level of coreceptor expression, it is equally plausible for selection to be determined by the relative permissiveness of the cell subset available for infection. The regions of the female genital tract most likely to be exposed to HIV-1 are protected by mucosal epithelia [8,10,43-47]. There is little compelling evidence that epithelial cells can be infected or that they can transfer HIV or HIV-infected cells from the surface of the epithelia to permissive cells lying in the submucosa by transcytosis [46] (reviewed in [37]). Surrogate models of transmission have shown that T cells and macrophages located in the submucosal layer are the first cells in which HIV infection can be detected [40,46,48]. How the virus is able to infect permissive cells located beneath the epithelial layer remains unclear, although the most likely routes are via physical damage and direct breach of the epithelial barrier or direct infection of interdigitating intra-epithelial cells such as macrophages (reviewed in [37]). Infection of macrophages is most efficient for R5 and dual-tropic isolates even though they express CXCR4 [49,50]. Therefore, preferential transmission of R5 might arise through an increased frequency of macrophages within the genital epithelia. Another possibility is that the apparent favouring of R5 variants is determined by the pool of variants within the genital tract of the transmitter. This hypothesis is supported by known transmission events involving CXCR4-tropic (X4) variants [24,51].
Finally, we have also shown that CCR5 chemokine mRNAs were expressed over a broad range in both the female and male genital tract and that expression levels were in good agreement with other reports [13,52]. In both sexes, SDF-1 mRNA was not detected, which is not surprising as SDF-1 is restricted to the basal ectocervical epithelia [53]. The effects of chemokine expression on HIV infection are hard to delineate, given that on one hand their presence will attract HIV susceptible cells to the genital mucosa yet on the other compete with HIV for cellular binding and down-regulate cell surface coreceptor presentation [48,54,55].
In summary, we have shown that expression of the CXCR4 and CCR3 coreceptors in regions of the male and female genital tract are significantly higher than that observed for CCR5. Consequently, we argue that coreceptor expression alone cannot account for the apparent discrepant frequency of transmission of R5 and X4 isolates.
Acknowledgements
The authors thank Sue Bainbridge for technical assistance and Dr Anna Grabowska for provision of liver cDNA. This work was funded by AVERT, Registered Charity Number 1074849, and by grants from the Nottingham University Hospitals Special Trustees.
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