Chronic airway inflammation provides a unique environment for B cell activation and antibody production.

BACKGROUND: B cells play many roles in health and disease. However, little is known about the mechanisms that drive B cell responses in the airways, especially in humans. Chronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10% of Europeans and Americans. A subset of CRS patients develop nasal polyps (NPs), which are characterized by type 2 inflammation, eosinophils and group 2 innate lymphoid cells (ILC2s). We have reported that NP contain elevated levels of B cells and antibodies, making NP an ideal system for studying B cells in the airways. OBJECTIVE: We sought to determine the mechanisms that drive B cell activation and antibody production during chronic airway inflammation. METHODS: We analysed B cells from NP or tonsil, or after ILC2 coculture, by flow cytometry. Antibody production from tissue was measured using Luminex assays and the frequency of antibody-secreting cells by ELISpot. Formation of B cell clusters was assessed using immunohistochemistry. Expression of genes associated with B cell activation and class switch recombination was measured by qRT-PCR. RESULTS: NP contained significantly elevated frequencies of plasmablasts, especially those that expressed the extrafollicular marker Epstein-Barr virus-induced protein 2 (EBI2), but significantly fewer germinal centre (GC) B cells compared with tonsil. Antibody production and the frequency of antibody-secreting cells were significantly elevated in NP, and there was evidence for local class switch recombination in NP. Finally, ILC2s directly induced EBI2 expression on B cells in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest there is a unique B cell activation environment within NP that is distinct from classic GC-mediated mechanisms. We show for the first time that ILC2s directly induce EBI2 expression on B cells, indicating that ILC2s may play an important role in B cell responses. B cell-targeted therapies may provide new treatment options for CRSwNP.

Differential growth rates in stromal cultures of human prostate derived from patients of varying ages.

BACKGROUND: This study was undertaken to attempt to characterize changes in in vitro growth rates and cellular phenotypes of human prostatic stroma associated with aging and/or development of benign prostatic hyperplasia (BPH). METHODS: Prostate stromal cell strains were established from 12 tissue donors of varying age. Culture growth rate was determined by cell counts over a 6-day period. Cell phenotype was assessed by immunocytochemical staining for smooth muscle alpha-actin, smooth muscle myosin, and prolyl-4-hydroxylase. RESULTS: Growth rates of prostate stromal strains in vitro varied. Stromal cells derived from aged males with BPH had significantly slower growth rates than cells from younger donors. A positive reaction for prolyl-4-hydroxylase, a mesenchymal cell marker, was present in all cell cultures regardless of donor age. Expression of smooth muscle-specific actin, a nonspecific smooth muscle cell marker, was present in 48-79% of prostate stromal cultures. Staining for smooth muscle myosin, a specific smooth muscle cell marker, was found to vary significantly with age. The percentage of smooth muscle myosin-positive cells derived from males aged 15, 45, 57, and 72 years were 0.70 +/- 0.14%, 2.12 +/- 0.30%, 4.20 +/- 0.89%, and 26.25 +/- 1.0%, respectively. The shape and size of actin- and/or myosin-positive stromal cells from a 72-year-old donor culture were also usually larger and polygonal in shape as compared to thin and elongated shapes in 15-year-old donor cultures. The shape of actin- and/or myosin-positive cells from a 45-year-old donor culture demonstrated both phenotypes. CONCLUSIONS: These results suggest that in human prostate stromal cells cultured as described, the growth rate decreases, the percent of smooth muscle cells increases, and the cellular shape changes with increasing donor age and/or development of BPH.

Synergistic action of steroids and spermatocele fluid on in vitro proliferation of prostate stroma.

PURPOSE: Our goal is to understand human prostate growth phenomena potentially important to BPH development and growth. The objective of the present study is to characterize in vitro prostate stromal proliferative factors in testis epididymal secretions. MATERIALS AND METHODS: Human spermatocele fluids were used as a source of testicular epididymal plasma (STEP). Primary cultures of human prostate stromal cells were routinely grown in RPMI-1640 with 10% fetal bovine serum. During a 6-day experimental period, cells were cultured in RPMI-1640 in the absence of serum but supplemented with ITS. Whole STEP, ether stripped STEP, or heparin affinity column treated STEP was included in the culture medium with and without the addition of testosterone (T), dihydrotestosterone (DHT), or estradiol (E). Results of these treatments were assessed by cell counts. Antibodies against smooth muscle myosin heavy chain, smooth muscle alpha actin, and prolyl-4-hydroxylase were utilized in immunocytochemical characterization of cultured cells. RESULTS: Whole STEP stimulated prostatic stromal cells derived from prostates of 15, 45, 70 and 72-year-old men. Treatment of STEP by ether stripping or heparin affinity column exposure did not result in a significant reduction in cell counts. With the exception of the 15-year-old specimen, addition of T or DHT to ether stripped STEP resulted in a significant increase in cell counts over that of ether stripped STEP treatment alone. Preliminary immunocytochemical evaluation indicated the presence of variable mixture of fibroblasts, myofibroblasts, and smooth muscle cells in these cultures. CONCLUSIONS: These in vitro observations indicate that testis epididymal secretions contain androgen/STEP synergistic and androgen independent STEP factors promoting prostate stromal growth. These factors are not heparin binding. These observations are consistent with the concept that, in addition to the production of steroids, the testis produces non-androgenic factors that act in concert with, as well as independently of, androgen to stimulate prostatic growth.

Effect of spermatocele fluid on growth of human prostatic cells in culture.

The present study was carried out to investigate whether testicular fluid derived from a spermatocele contains substance(s) that promote the growth of human prostatic cells in culture. Human spermatocele fluid was centrifuged to sediment spermatozoa. The supernatant was then added to cultures of human prostatic stromal or epithelial cells that were isolated from surgical specimens of benign prostatic hyperplasia. Addition of spermatocele fluid in quantities of 1 microgram/ml of protein resulted in a significant increase in the number of both prostatic stromal and epithelial cells at the end of a 6-day culture period. Human serum at equivalent protein concentrations in the culture medium had no stimulatory effect. At least two separate growth-promoting factors were found in spermatocele fluid, one for stromal cells and one for epithelial cells. The mitogen for stromal cells was heat labile and persisted after treatment with activated charcoal. The factor for epithelial cells was heat stable but was removed by charcoal treatment. These observations are consistent with the concept that the human testis secretes nonandrogenic substances that can promote prostatic growth.