Clinical studies suggested thatandrogen might be associated with infiltrating T cells in prostate of benign prostatic hyperplasia (BPH) patients, but detail of T-cell subset and mechanism still remained unclear. The present study tested the hypothesis that intraprostatic 5
Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in aging men [
To date, chronic inflammation has been recognized as another key player in the BPH pathogenesis and progression [
The most important potential cause for immune response in prostate is the prostatic microenvironment [
In the present work, we focused on the relationship between intraprostatic DHT level, BPH epithelial cells, and T-cell infiltration. We further elucidated the chemokine changes in different level of intraprostatic DHT.
The patients were selected by considering medication duration from the medical records of 726 patients who underwent transurethral resection of the prostate (TURP) between January 2008 and December 2011 in Peking University First Hospital. According to the medication history, prostate tissue was obtained from 64 prostatic hyperplasia patients by transurethral resection. Patients were divided into two groups: group 1 consisted of 28 patients who had been medicated neither with
Monoclonal anti-CCL5 antibodies and the recombinant protein IgG were purchased from R&D systems (MAB678, MAB002, Minneapolis, MN, USA), and 500
Blood samples were collected in sterile heparinized containers from 6 health persons at 10 mL per tube. Purification of human Peripheral blood mononuclear cells was performed according to some previous publications [
Prostate tissue preparation was performed as the publication description [
The BPH epithelial cell-line, BPH-1, was purchased from KeyGen Biotech Co., Ltd (KG1008, NJ, China) and the predominantly CD8(+) T-lymphocytic cell-line, Molt-3 [
To determine the expression of CD4 and CD8 on the cell surface and intracellular CCL5 in BPH tissues, the specimens from TUR-P surgery were fixed in 4% buffered formalin overnight at 4°C and then dehydrated in an ascending ethanol series, routinely embedded in paraffin, and sectioned at 3
The max density of CD4 positive or CD8 positive cell was defined as ratio between the maximum positive cell number and all of nuclear cell number under 100x field. And these indexes were average value by two operators who are blind to each other and were calculated with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Proteins expression of CCL5 was assessed semiquantitatively and a 4-tiered system (0 negative, 1 weak, 2 moderate, and 3 strong) was used. Two pathologists evaluated the stain strength and the final result was the average of their scores.
To further study the T-cell subpopulation infiltration among the total T cell, the isolated lymphocytes from prostate tissues were analyzed by two-color flow cytometry for phenotypic characterization of T cells according to manufacturer’s procedure. Cells were resuspended in staining buffer (PBS containing 1% fetal bovine serum) and stained for 30 min at 4°C with anti-CD3 PE-Cy 7 and anti-CD4 FITC or anti-CD8 PE and their isotype control antibodies (both from BD Biosciences). Flow cytometry was done on a Becton Dickinson LSRII (BD Biosciences);
Total RNA was extracted from each cell line using Trizol (15596-018, Invitrogen, Grand Island, NY, USA). According to manufacturer’s protocol, cDNA was synthesized from 1
The sequences of q-PCR primers.
Gene | Forward primer | Reverse primer | Size | Number |
---|---|---|---|---|
CCL2 | AGCAAGTGTCCCAAAGAAGC | CATGGAATCCTGAACCCACT | 93 | NM_002982 |
CCL5 | ACCACTCCCTGCTGCTTTG | ACACTTGGCGGTTCCTTCG | 212 | NM_001278736 |
CCL8 | ATGCTGAAGCTCACACCCTT | TCAAGCTCTGACTCTCAGTCCA | 363 | NM_005623 |
CCL15 | ATATAATAATAAAGAGACAAAAGAGGC | TACTCTTTATTAGATGCATTACTTTCA | 134 | NM_032965 |
CCL19 | AGCTCCTCTGCACCAGACCT | TAGTTGTAAACACCAGGCGG | 150 | NM_006274 |
CCL21 | GATGCAGCGTCTGGACAA | TTGGAGCCCTTTCCCTTC | 102 | NM_002989.3 |
CCL23 | TGTGTCCAGCTTCAGCATTC | TTTGAAACGAACAGCGAGTG | 126 | NM_145898 |
CCL28 | AGAAGCCATACTTCCCATTGC | AGCTTGCACTTTCATCCACTG | 208 | NM_148672 |
CXCL1 | AGGGAATTCACCCCAAGAAC | CACCAGTGAGCTTCCTCCTC | 204 | NM_001511 |
CXCL2 | CTGCCCTTACAGGAACAGAA | ATCAGGATTGAACTAACTTGGG | 250 | NM_002089 |
CXCL8 | ACCGGAAGGAACCATCTCACT | ATCAGGAAGGCTGCCAAGAG | 75 | NM_000584 |
CXCL9 | ATTGGTGCCCAGTTAGCC | CATCAGCAGTGTGAGCAGTG | 143 | NM_002416 |
CXCL10 | GCTGCTACTACTCCTGTAGGAAGG | TGGAAGATGGGAAAGGTGAG | 159 | NM_001565 |
CXCL11 | ATGAGTGTGAAGGGCATGGC | TCACTGCTTTTACCCCAGGG | 121 | NM_005409 |
CCR1 | TCAACAAAGTCACCCACTTCC | GTGTCTCCCATGGCTTAGGA | 106 | NM_001295 |
CCR3 | TGACTGTGAGCGGAGC | ATGTATCTGCCCAGGTGC | 171 | NM_178329 |
CCR5 | GACTCTTGGGATGACGC | GATCGGGTGTAAACTGAGC | 177 | NM_000579 |
CXCR2 | ACATTCCAAGCCTCATGTCC | CTTAGAACATAGAGTGCCATGGG | 217 | NM_001168298 |
CXCR4 | ACGTAAAGCTAGAAATGATCCCC | GTACACTGTAGGTGCTGAAATCAAC | 190 | NM_003467 |
To detect the recruitment of CD8+ T cell by tissue lysates and BPH-1 cells, the tissue lysates or
Statistical analyses for continuous variables involved paired
We detected T-cell population infiltration between prostate tissue with/ without finasteride treatment [
The T-cell population infiltrating prostate tissue with/without finasteride treatment. (a) CD8 was stained from no medication group and finasteride group; scale bar: 100
Furthermore, as shown in Figure
To study the potential cross talk between infiltrating CD8+ T cell and prostate epithelial cells as seen in BPH specimens (Figure
CD8+ T cells migration
This data was then confirmed in BPH epithelial cell-line. As shown in Figure
The q-PCR was used to assay for the most reported chemokines that are related to attracting T cells [
Induction of chemokines in BPH-1 cells stimulated by changes of DHT level. (a) Q-PCR screening of a panel of cytokine factors that could be responsible for BPH-1 cell promoted T-cell migration. Compared to the BPH-1 cells cultured with normal medium, mRNA transcription of CCL5 was upregulated in BPH-1 cells with charcoal medium treatment;
Next, interruption assay was detected by using CCL5 neutralizing antibody in the migration system. It was shown that blocking CCL5 led to significantly suppressing the Molt-3 cells migration toward BPH-1 cells in low DHT condition Figure
The CCL5 expression was investigated in above BPH patients by IHC staining as shown in Figure
CCL5 immunolocalization in prostate tissue samples by IHC. (a) CCL5 was stained from no medication group and finasteride group. Magnification and negative control are the same as mentioned before. (b) The average immunoreactive score of CCL5 in different group was quantified (mean ± SEM).
At present, so many studies have shown the role of chronic inflammation in BPH development. Cytokines, growth factors like IL6, IL8, IFN-r produced by T-lymphocytes, and BPH cells are involved in altering tissue remodeling and hyperplastic growth at each stage of BPH [
It is reported that prostatic immune inflammatory cells consist of 70% T lymphocytes, 15% B-lymphocytes, and 15% macrophages, as well as mast cells [
The IHC results in this study showed CD8+ T cells localized surrounding epithelial area in BPH tissue. BPH epithelial is an important component of microenvironment in the prostate tissue. It can acquire the ability to express class II MHC molecules [
Results in this study demonstrated that intraprostatic DHT has strong immune suppressive effects on CD8+ T cell infiltration induced by BPH epithelial cells. Some other groups have also discussed the similar study. Vignozzi et al. highlighted that DHT exerts an immune regulatory role on human prostatic stromal cells, inhibiting their potential to actively induce and/or sustain autoimmune and inflammatory responses [
To further dissect how BPH-1 recruited more Molt-3 cells, we applied q-PCR to examine the expression of T cell related chemokines in BPH-1 cells at low DHT level. We found that CCL5 which functions as a chemokine playing a critical role in the recruitment of T cells [
Then CCL5 expression was identified in the clinical sample mentioned above. Higher expression of CCL5 is showed in BPH tissues after finasteride treatment by immunohistochemistry. Importantly, the CCL5 was localized surrounding the epithelial area. In normal and BPH prostate tissue, infiltrating CD8+ T cells are mainly localized around epithelial ducts [
In conclusion, the most striking finding of the present study is that DHT exerts an immune regulatory role on human prostate epithelial cell, inhibiting their potential to actively induce inflammatory responses, and CCL5 which secreted by prostate epithelial cell is the key chemokine in this progression (Figure
Mechanism and regulatory pathway of low intraprostatic DHT-promoted CD8+ T cell infiltration in BPH prostate tissue. Low intraprostatic DHT level could promote BPH epithelial cells to recruit more CD8+ T cells via upregulation of CCL5 mRNA transcription.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Yu Fan and Shuai Hu contributed equally to this work.
This work was supported by National Natural Science Foundation of China grants 81370858. The authors also thank Department of Urologic Pathology, Peking University First Hospital, for samples support.