Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis

<div><p>Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in <i>HPS1</i> which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the <i>HPS1</i> mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients’ serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with <i>HPS1</i> rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by <i>HPS1</i> transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.</p></div

Results of transfection of HPM cells with normal <i>HPS1</i>.

<p>A) Representative Western blot of <i>HPS1</i> transfected HPM cells and control LAD2 cells demonstrating HPS-1 protein expression (arrow) in 16 hr, 2 and 4 wk HPM cultures, confirming transfection and expression of HPS-1 protein. Immunoblot is representative of at least 2 independent experiments; B) Slowing of doubling time 5–7 fold immediately following transfection with <i>HPS1</i>. Transfected cells reached average doubling rate of 3–4 days by 10 wks; C) FSC and SSC changes in HPM cells 4 wks following transfection. No additional changes were noted following supplementation of cultures for 4 wks with rhSCF and rhIL-6. Data are representative of 2 separate experiments performed in duplicate; D) Three-fold increase in cellular β-Hex content following transfection (upper graph) and ten-fold increase in cellular histamine content 4 wks following transfection (lower graph). Data are the means ± SEM (n = 3). **p<0.01.</p

Characteristics of HPM cells.

<p>A) Representative dot plots of HPM clones show that HPM cells are FcɛRI+/CD117^low/-; B) Histamine content of HPM clones ranges from 0.1–0.3 pg/cell; C) Representative flow cytometry from clone #4 of permeabilized HPM cells in the presence (blue line) or absence (yellow line) of rhSCF and rhIL-6 shows similar expression of tryptase, chymase and carboxypeptidase; D) β-Hex release (upper graph) of HPM clones #3 and #4 at 1 ng/ml antigen is minimum and ranged from 3–5% following crosslinking with antigen. In the presence of 0.1 ng/ml Ag, the addition of increasing concentrations of SCF does not enhance release (lower graph) from either clone. Data are from 2 separate experiments performed in duplicate (A-D); E) Thapsigargin but not FcɛɛRI crosslinking with SA induces calcium influx in HPM clones; F) In contrast to low migration of normal (red column) and HPS-1 (white column) HuMCs to all concentrations of SCF, representative chemotaxis of HPM clone #4 shows HPM migrated cell numbers (black column) are significantly elevated and show increasing migration to increasing concentrations of SCF. Data are representative of 2 separate experiments performed in duplicate (E-F), and G) Representative light microscopy of extracellular matrix formation in HPM clones 3 and 4 (arrow–matrix).</p

Characteristics of normal and HPS-1 HuMCs.

<p>A) Wright-Giemsa staining of normal (left panel) and HPS-1 (right panel) HuMCs. HPS-1 granules exhibit less dense staining. Representative flow cytometry of size (FSC) and granularity (SSC) of HuMCs. Grey-shaded histograms are normal HuMCs, green lines represent HPS-1 HuMCs, blue-shaded histograms are isotype controls; B) Representative flow cytometry of CD surface marker histograms of HuMCs. Grey-shaded histograms are normal HuMCs, green lines represent HPS-1 HuMCs, blue-shaded histograms are isotype controls. CD117 and FcɛRI expression of the CD117^high/FcɛRI+ population is reduced in HPS-1 HuMCs. CD63 and CD203c activation marker expression is increased; C) Representative flow cytometry of permeabilized HPS-1 HuMCs (histograms) shows tryptase and carboxypeptidase staining is somewhat increased in HPS-1 HuMCs but not chymase staining. Grey-shaded histograms are normal HuMCs, green lines represent HPS-1 HuMCs, blue-shaded histograms are isotype controls. HPS-1 HuMC carboxypeptidase but not tryptase content (bar graphs) is increased based on protein assays; data are the means ± SEM (n = 3). *p<0.05. Histograms (A-C) are representative of at least 3 separate experiments; D) Histamine content (pg/cell) of HPS-1 HuMCs is reduced when compared with normal HuMCs; E) β-Hex release (upper graph) of HPS-1 HuMCs was reduced at higher concentrations (>1 ng/ml) of antigen when compared with normal HuMCs. β-Hex release (lower graph) was enhanced but at reduced levels for HPS-1 HuMCs in the presence of 0.1 ng/ml antigen with increasing concentrations of SCF. Data are the means ± SEM (n = 3) (D-E). *p<0.05, **p<0.01; F) Representative flow cytometry of CD117 and annexin V expression of normal and HPS-1 HuMCs incubated with imatinib for 24 hrs (left-sided histograms) and 5 days (right-sided histograms). Red lines are untreated HuMCs, blue lines are imatinib-treated cells, blue-shaded histograms are isotype controls. Histograms are representative of 2 separate experiments performed in duplicate.</p

Differential gene expression and statistically significant networks.

<p>A) Heat map display of the strongest, differentially expressed genes measured in HPS-1 HuMCs compared with normal HuMCs; B) Heat map display of mock transduced (control) versus <i>HPS1</i> transduced HPM cells showing significant differentially expressed and enriched genes; C) FN-1 is a master regulator of amyloid precursor protein APP, CASP7, CD79A and ZBTB7A. Target molecules in the dataset found for APP were CTSB, CTSV, IL-1A and IL-6; D) Central to the FN-1 and APP relationship (left-sided diagram) is a network that in its turn targets the IL-6 network (right-sided diagram), all of which are significantly upregulated. Genes are represented in the nodes and gene-gene relationships in the edges. The intensity/color is a measure of the extent of differential expression. The shapes of nodes represent categories of gene function; FN-1 is an enzyme and shown as a diamond, IL-6 is a cytokine and shown as a square. IL-6 operates in the context of KIT, STAT3, SOCS1, GATA3 and CAMP.</p

Pulmonary mast cells localize to fibrotic regions of the lung parenchyma in HPS-1 pulmonary fibrosis. Histology, immunohistochemistry and ultrastructure of HPS-1 derived dermal mast cells.

<p>A) Representative anti-tryptase stained lung sections from patients with HPS-1 pulmonary fibrosis and IPF showed significant numbers of pulmonary mast cells within fibrotic alveolar interstitium. Mast cells are shown in normal lung tissue for comparison. Data are representative of four separate patients with either HPS-1 or IPF; B) Staining of skin biopsies reveals normal numbers of melanocytes (Melan-A) (upper panels) but decreased melanin pigmentation (Fontana-Masson) in HPS-1 patients (lower right panel; compare with lower left panel); (C) Wright-Giemsa staining (upper panels) and tryptase staining (lower panels) of dermal mast cells; (D) Ultrastructure of normal and HPS-1 HuMC granules used for classification include dense patches, cores, mottling, dense fill, less dense fill and extracellular patterns, with or without scrolls or lattices, and E) Comparison of normal and HPS-1 granules showed dense patches with scrolls were significantly increased in normal mast cell granules as compared with less dense fill increased in HPS-1 granules, when granules were scored by three independent observers blinded to the identity of originating mast cells. Data are the means ± SEM (n = 3). *p < 0.05, **p < 0.01.</p

Demographics of HPS-1 patients.

<p>Demographics of HPS-1 patients.</p

Functional groups enriched significantly in cultured HPS-1 HuMCs and non-transduced HPM cells.^*

<p>Functional groups enriched significantly in cultured HPS-1 HuMCs and non-transduced HPM cells.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159177#t003fn001" target="_blank">*</a>}</p