All women underwent careful gynecologic and laboratory evaluation; results of the evaluation did not reveal concomitant infectious or gynecologic problems in any of the individuals enrolled in the study. All ESN, HIV-seropositive, and HC women had been longitudinally followed for at least 3 years (prior to the study period) by the Department of Obstetrics and Gynecology of Santa Maria Annunziata Hospital in Florence. This allowed us to exclude from the study ESNs and HCs in whom sexually transmitted diseases or any other tumoral or gynecologic pathology had been reported in that period.
The ESNs were characterized on the basis of the presence of CCR5-Δ32 alleles; a heterozygous deletion was detected in 1 individual. All ESNs, HIV patients, and low-risk uninfected women agreed to donate peripheral blood mononuclear cells (PBMCs) and to undergo multiple cervical biopsies. Three different bioptic mucosal specimens were collected in all the women who participated in the study: 1 specimen was from the anterior fornex, 1 from the posterior fornex, and 1 from the cervix. Bioptic tissues were placed in a nalgene vial containing 1 mL of RPMI (Roswell Park Memorial Institute) (Organon Teknika Corp., Durham, NC) or embedded in OCT and immediately deep-frozen in a liquid nitrogen tank.
Sample Collection and Processing
Whole blood was collected by venopuncture in EDTA-containing vacutainer tubes (Becton Dickinson & Co., Rutherford, NJ). PBMCs were separated on lymphocyte separation medium (Organon Teknika Corp.) and washed twice in phosphate-buffered saline (PBS), and the number of viable leukocytes was determined by trypan blue exclusion. Cervical samples were obtained, as described elsewhere, 3 by using a cytobrush (Histobrush; Spectrum Labs, Dallas, TX), which was inserted in the cervical os and rotated 360°, and the sample thus obtained was transferred immediately into 2 mL of RPMI. The cytobrush specimen was not collected from menstruating women and was not used if it contained visible blood. The cytobrush was agitated and discarded, and the remaining cell suspension was once more agitated to loosen clumps of mucosa. Cervicovaginal mononuclear cells (CVMCs) were then isolated according to the protocol used for PBMCs and were resuspended in RPMI. The mean (median) (± SD) number of CVMC cells obtained was 200,769 (131,000) (± 56,678).
CD8+ T-Lymphocyte Purification
PBMCs were incubated for 1 hour in RPMI + 20% AB serum in plastic flask to remove monocytes and dendritic cells. Monocyte-depleted cells were incubated with 20 μL of MACS CD8 Microbeads (Miltenyi Biotec, Auburn, CA) for 15 minutes at 4°C. Magnetic separation with positive selection columns was performed using a miniMACS separator. The purity of the cells obtained was >99% as judged by flow cytometry.
Production of α Defensin
1 × 106 purified CD8+ T cells or 5 × 105 CVMCs were incubated in the presence/absence of α CD3 (0.1 μg/m) (Serotec, Oxford, UK) and α CD28 (5 μg/mL) (R&D Systems, Minneapolis, MN) antibodies, together with an equal number of allogenic irradiated PBMCs and 20 U/mL of recombinant interleukin-2 (IL-2). After 3 days of incubation at 37°C in a humidified CO2 incubator, supernatants were collected by centrifugation and cells were analyzed for α defensin production by enzyme-linked immunosorbent assay (ELISA) (Hbt Human HNP 1–3, HyCult Biotechnology; Uden, The Netherlands) (the antibodies used in the ELISA assay recognize human α defensins 1, 2, 3), following the procedures suggested by the manufacturer.
Flow Cytometric Analysis for α Defensin
Peripheral CD8+ cells and CVMCs were washed in PBS and stained with a CD8-specific monoclonal antibody coupled to R-phycoerythrin-Cyanine 5 Tandem (TC) (mouse IgG2a isotype, Caltag Laboratories, Inc., Burlingame, CA) for 30 minutes at 4°C in the dark. Cells were then washed and fixed in Reagent A solution (FIX & PERM cell permabilization kits; Caltag Laboratories) for 10 minutes at room temperature in the dark. The cells were washed once again in PBS and re-suspended in Reagent B (FIX & PERM cell permabilization kits) with an α defensin-specific monoclonal antibody (D21; HyCult Biotechnology) (this antibody recognizes human α defensin 1, 2, and 3) followed by fluorescence labeling with streptavidin-FITC (Caltag Laboratories). The cells were then fixed in 1% paraformaldehyde in PBS. Cytometric analyses were performed using an EPICS XL flow cytometer (Beckman-Coulter, Inc., Miami, FL) equipped with a single 15-mW argon ion laser operating at 488 nm interfaced with 486 DX2 IBM computer (IBM, Cambridge, UK). For each analysis, 20,000 events were acquired and gated on CD8 expression, and side scatter properties. For CVMC samples, only 2000 events were acquired due to the low number of cells. Green fluorescence from FITC (FL1) was collected through 525-nm bandpass filter and deep-red fluorescence from TC (FL4) was collected through 670-nm bandpass filter. Data were collected using linear amplifiers for forward and side scatter and logarithmic amplifiers for FL1 and FL4. Samples were first run using isotype control or single fluorochrome-stained preparations for color compensation. Representative data obtained with the isotype control antibody are shown in Figure 1.
RNA was extracted from PBMCs or from cervical biopsies with the acid guanidium thiocyanate-phenol-chloroform method. Purity was determined by spectrophotometry. RNA was finally treated with RNase-free DNase (RQ1 DNase, Promega, Madison, WI) to remove the contamination of genomic DNA.
One microgram of total RNA from cervical biopsies was reverse transcribed into first-strand cDNA in a 20-μL final volume containing 1 μM of random hexanucleotide primers, 1 μM of oligo dT, and 200 U of Molony murine leukemia virus reverse transcriptase (Promega).
Normalization of Sample GAPDH cDNA Content by Competitive PCR
To compare α defensin mRNA expression, clinical samples were normalized for GAPDH cDNA content by competitive polymerase chain reaction (PCR) (TaKaRa, Otsu, Japan). Briefly, 1 set of primers amplified both target GAPDH cDNA and competitor cDNA in each sample. The following primers were used (these primers are specific for human α defensin 1): forward:GCAAGAGCTGATGAGGTTGC reverse: GTTCCATAGCGACGTTCTCC. The PCR products of both target and competitor were subjected to acrylamide gel electrophoresis. The competitor cDNA generated a longer PCR product (241 bp) than the target cDNA (199 bp). Densitometry was used to quantify the density of the bands of target and competitor PCR products. The amount of substrate GAPDH cDNA in each sample was calculated plotting the ratio of sample density to competitor PCR product against the known amount of competitor substrate cDNA. All samples were diluted to the same concentration as the sample with the lowest GAPDH cDNA concentration. α Defensin mRNA expression was quantified using a Quantitative Competitive PCR Kit (TaKaRa). The results were analyzed as previously described for GAPDH quantification and expressed as α defensin/GAPDH ratio.
Immunochemistry for α Defensin Detection
Tissue samples from ESN, HIV-positive, and HC individuals were embedded in OCT, frozen in dry ice, and stored at −80°C. Using a cryostat maintained at −25°C, 6-mm sections were serially cut and transferred to poly-l-lysine-coated slides. These were air-dried, fixed in 2% paraformaldehyde (pH 7.4, Sigma-Aldrich) for 15 minutes, washed in PBS 1×, air-dried, and stored at −20°C until use. At least 30 sections were cut from each bioptic specimen.
In situ α Defensin Detection
Cryosections were air-dried and then incubated with 2% fetal calf serum (FCS) in balanced salt solution (BSS) (Gibco, Ltd., Paistey, OK) -saponin for 30 minutes at room temperature. The endogenous peroxidases were blocked by 1% H2O2/0.02 NaN3 in BSS, supplemented with 0.1% saponin for 30 minutes. Thereafter the avidin and the biotin in the tissue were blocked with the avidin for 15 minutes and with the biotin for 15 minutes, respectively. The sections were washed with BSS-saponin and then incubated overnight at room temperature with monoclonal biotinylated antibody specific for α defensin (D21) (1:500). This step was followed by incubation with horseradish streptavidin-biotin-coupled peroxidase followed by incubation with vectastain (Vector Laboratories, Burlingame, CA) for 30 minutes and by diaminobenzidine (DAB substrate KIT, Vector) and counterstained with hematoxylin and eosin to show morphology. All antibody reagents were appropriately diluted in 0.1% saponin containing BSS and the incubations were followed by rinses in 0.1% saponin containing BSS, except the steps following horseradish peroxidase-conjugated streptavidin, from which saponin is omitted. To test the specificity of the staining, immunohistochemistry was performed as previously described after the exclusion of the defensin monoclonal antibodies. The immunohistochemically stained cells were examined (5 different fields for each section) with a Leica (Wetzlar, Germany) DMR-X microscope (magnification 100×) and the quantification of individual α defensin-producing cells was performed by automated computerized image analysis. The experiments were conducted in a blinded fashion.
The statistical analysis was based on a nonparametric Jonckheere-Terpstra test for trends. All data were also analyzed by a nonparametric Kruskal-Wallis test. Comparisons between the different groups were made using a 2-tailed t-test. Possible relationships were evaluated using a Pearson correlation test. Statistical analysis was performed using the SPSS statistical package (SPSS, Inc. Chicago, IL).
α Defensin Production by CD8+ T Lymphocytes and Cervicovaginal Cells
Production of α defensin by peripheral blood CD8+ T lymphocytes and by CVMCs was examined in unstimulated cells and in cells stimulated with a combination of monoclonal antibodies (αCD3 and αCD28), cytokines (IL-2), and allogeneic cells, as previously described (because of limitations in the number of available cells, unstimulated production of α defensin could not be measured in CVMCs). Cells of 9 ESNs, 10 HIV-infected patients, and 13 low-risk HCs were analyzed. Table 1 summarizes the clinical and epidemiologic characterization of the 9 ESN women recruited in the study. In basal conditions (unstimulated cells), the highest production of α defensin was detected in peripheral blood CD8+ T lymphocytes of ESNs (median: ESN = 462.9 pg/mL; HIV = 308.7 pg/mL; low-risk controls = 45.6 pg/mL). Thus, a 10-fold difference was observed when α defensin production by ESNs was compared with that of low-risk controls; the amount of α defensin produced by lymphocytes of ESN and HIV patients was similar (ESN vs. low-risk HCs, P = 0. 001; HIV vs. low-risk HCs, P = 0. 02; ESN vs. HIV, P = NS). To exclude the possibility that positive bead selection could per se stimulate the production of α defensin, we measured the generation of these peptides by purified CD8 (3 HC and 3 HIV patients) that were positively or negatively selected. α Defensin production was comparable in both conditions (data not shown). Upon stimulation, α defensin production by peripheral blood CD8+ T lymphocytes of ESN and HIV patients augmented <3-fold; in contrast, a >10-fold increase was observed in lymphocytes of low-risk HCs (median: ESN = 1198.1 pg/mL; HIV = 1159.6 pg/mL; low-risk controls = 492.2 pg/mL). Statistical significance was nevertheless maintained (ESN vs. low-risk HCs, P = 0. 001; HIV vs. low-risk HCs, P = 0. 044; ESN vs. HIV, P = NS) (Fig. 2A). Stimulated production of α defensin by CVMCs was higher, albeit not significantly in ESNs compared with either HIV patients or low-risk HCs (Fig. 2B).
α Defensin-Producing CD8+ T Lymphocytes in Peripheral Blood and Cervicovaginal Lavages
α Defensin-producing CD8+ T lymphocytes were investigated using FACS analyses in peripheral blood and in cervicovaginal lavages of all ESN, HIV patients, and low-risk healthy women. Results showed a 10-fold increase of unstimulated peripheral blood CD8+, α defensin-producing lymphocytes in ESN compared with low-risk HCs; these cells were not significantly different in ESN and HIV patients (ESN vs. low-risk HCs, P = 0. 003; HIV vs. low-risk HCs, P = 0. 01; ESN vs. HIV, P = NS) (Fig. 3). In αCD3+αCD28+IL-2+allogeneic cell–stimulated cultures, CD8+ α defensin-producing lymphocytes were increased only marginally in ESNs, were slightly decreased in HIV patients, but, once again, showed a >10-fold increase in low-risk HCs (ESN vs. low-risk HCs, P = NS; HIV vs. low-risk HCs, P = 0. 006; ESN vs. HIV, P = 0. 01).
The highest percentage of α defensin-producing CD8+ T lymphocytes was seen in cervicovaginal lavages of ESNs; the differences between HIV patients and low-risk HCs were not statistically significant whereas the number of these lymphocytes was significantly increased in ESNs compared with low-risk HCs (P = 0. 01). Representative results are shown in Figure 3; the data are summarized in Table 3.
α Defensin mRNA in Peripheral Blood Mononuclear Cells and in Cervicovaginal Biopsies
α Defensin mRNA was analyzed in PBMCs and in the genital mucosal biopsies of CD8+ T lymphocytes of all ESNs, of HIV patients, and low-risk HCs. Results stemming from analysis performed in (unstimulated) PBMCs indicated that α defensin-specific mRNA is augmented in cells of ESNs compared with HIV patients and low-risk HCs (median α defensin/GAPDH ratio: ESN = 21.95; HIV = 8.70: HC = 12.72). These differences did not reach statistical significance; representative results are presented in Figure 4 (upper panels). Results obtained upon analysis of mRNA for α defensin in genital mucosal biopsies showed that α defensin-specific mRNA is augmented in ESNs compared with HIV patients and low-risk HCs (median α defensin/GAPDH ratio: ESN = 35.45; HIV = 17.98: HC = 3.37) (ESN vs. low-risk HCs, P = 0.03; HIV vs. low-risk HCs, P = 0.02; ESN vs. HIV, P = NS). Representative results are shown in Figure 4 (lower panels).
In Situ α Defensin Detection
α Defensins were analyzed immunohistochemically in bioptic samples from all ESNs, HIV patients, and low-risk healthy women. Results showed a higher signal in ESNs, intermediate in HIV-positive patients, and no signal in HCs. This pattern was detected in bioptic samples of all ESNs, 8/10 HIV patients (the remaining 2 patients had a pattern similar to that seen in ESNs), and all 13 low-risk healthy women. A positive staining was observed at the level of cervical epithelium and stroma, in particular in neutrophils and lymphocytes. In this first stage of analysis, only qualitative information was obtained. Representative results are presented in Figure 5.
Correlations Between α Defensin in the ESN and Viroimmunologic Data of the HIV-Seropositive Partners
α Defensin production by αCD3+αCD28+IL-2+allogeneic cell–stimulated peripheral CD8+ T lymphocytes of ESNs was negatively correlated with the absolute CD4 counts (r = −0.824; P = 0.012) and with the CD4% (r = 0.856; P = 0.007) seen in the HIV-infected sexual partner. Analogously, α defensin production by stimulated CVMC CD8+ T lymphocytes of ESNs was negatively correlated with the absolute CD4 counts (r = −0.721; P = 0.042) and with CD4% (r = −0.928; P = 0.001) of the HIV-infected sexual partner. A correlation (r = 0.606; P = 0.071) was also observed between the unstimulated production of α defensin by peripheral CD8+ T lymphocytes and HIV plasma viremia in the infected sexual partner. Finally, α defensin production by peripheral CD8+ T lymphocytes and by CVMCs of ESNs was strongly correlated both in unstimulated (r = 0.935; P < 0.001) and stimulated (r = 0.919; P < 0.001) cell cultures.
Defensins are a family of cationic peptides contained in a number of different immune cells, strictly conserved throughout phylogeny, and characterized by a potent antimicrobial activity. 23–26 A recent report showed that α defensins are also capable of inhibiting the infection of host cells by HIV and that the production of these peptides is higher in CD8+ T lymphocytes of HIV-infected LTNPs. 21
LTNPs are HIV-infected patients in whom disease progression is not observed; LTNPs have been extensively examined in the attempt to clarify the immune mechanisms associated with this condition. 27–29 Another important group of people that has been the subject of many investigations, and may be naturally resistant to HIV infection, is individuals who, despite multiple, continuous exposures to HIV, do not sero-convert or show any sign of disease (ESNs). 1,2 Immune surrogate markers of resistance to HIV infection in ESNs mainly include cells belonging to adaptive immunity: HIV-specific T 30–34 and B 3–7 lymphocytes. To verify whether the concentration of α defensin is increased in ESNs we analyzed, using an array of methods, mucosal and systemic α defensin in 9 HIV-uninfected sexual partners of HIV-infected individuals. Results demonstrate that the number of α defensin-producing CD8+ T lymphocytes is augmented both in the genital mucosa and in the peripheral blood of ESNs, and that α defensin mRNA is also increased in PBMCs and in cervical biopsies of ESNs. These results are the first to show that α defensins are augmented in these individuals. Because α defensins inhibit in vitro the infection of host cells by HIV, it is tempting to speculate that the increased concentration of these molecules seen in exposed seronegative individuals could contribute to their apparent resistance to HIV infection.
One of the most striking findings reported herein is that the production of α defensin, as well as the number of α defensin-producing cells, was 10-fold higher in ESNs than in low-risk HIV-unexposed controls, in cells that were not stimulated in vitro. This finding may be due to the fact that ESNs are repeatedly exposed to HIV and may therefore be an index of immune activation, or it could be genetically predetermined (i.e., a genetically determined potent production of α defensin is associated with the ESN status). The observations that no correlation was seen between α defensin and the date of the last at-risk sexual exposure to HIV (1 day–7 months prior to the study period), and that α defensins were increased both in ESNs and in HIV- patients, seem to offer support to the hypothesis that the increased α defensin production described herewith is a reflection of the chronic immune stimulation detected in ESNs and in HIV-infected patients. 35 Conversely, the fact that a higher production of α defensin was detected in the sexual partners of the HIV patients with lower CD4 counts, and thus more likely to transmit HIV infection, 36,37 might indicate that such production is modulated in response to the immunologic status of the contact case.
The maintenance of a high production of α defensin independently of antigenic stimulation (in this case, exposure to HIV) is a characteristic of mechanisms belonging to innate immunity 38,39 and is in contrast with a number of observations showing that such exposure is necessary to maintain HIV-specific T and B lymphocyte in ESNs. Thus HIV-specific T-helper and cytotoxic T-lymphocyte responses disappear within months after cessation of exposure to the virus in uninfected newborns of HIV-seropositive women 31 and in health care workers reporting a single professional exposure to HIV-seropositive body fluids 30; and the concentration of HIV-specific plasma IgA is significantly diminished in ESN women who adopt safe sex procedures. 3 It will also be interesting to verify why the percentage of a defensin-producing CD8+ T cells is lower, upon stimulation, in HIV patients compared with the other groups of individuals. Longitudinal studies, or the analysis of patients in different phases of disease, could explain whether this phenomenon is secondary to exhaustion or rather is associated with other yet unknown factors. Finally, it is important to underline that the observation of a robust production of α defensin by in vitro unstimulated cells argues against the possible identity between these peptides and CAF. Thus, CAF, the CD8− produced antiviral factor that, although still elusive, has originated all the research performed on soluble antiviral factors in HIV infection, is only produced by CD8+ T lymphocytes that have been stimulated in vitro, in antiviral treated patients 40 as well as in LTNPs and in ESNs. 16,17,41 In this light, recent data have definitively clarified that the biologic action of CAF is distinct from the one of α defensin. Thus, CAF inhibits HIV RNA transcription at the level of the long terminal repeat–driven gene expression, a process that is not influenced by α defensin. 22,42 One unresolved question is whether CD8 T lymphocytes are capable of supporting a robust production of α defensin. Data reported by Zhang et al. 21 and D’Agostino et al., 43 and the results presented herewith, favor such possibility, which is denied in an elegant report by Mackewicz et al. 42; more analysis will be needed to clarify this issue.
The fact that both the unstimulated and the stimulated production of α defensin and the numbers of α defensin-producing cells were similar in ESNs and in HIV-infected patients suggests that high α defensin production alone is not sufficient to confer resistance to HIV infection. Such resistance may stem from a still unqualified interaction between innate and adaptive immunity, genetic factors, and possibly other mechanisms that are still unresolved.
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Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
α defensin; T lymphocytes; HIV; immunology; exposed-uninfected individuals