CADM1 is a key receptor mediating human mast cell adhesion to human lung fibroblasts and airway smooth muscle cells

Abstract

Background: Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.

Objective: In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.

Methods: CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.

Results: Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.

Conclusion and clinical relevance: Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.

Figure 1. Surface expression of CADM1 in HLMCs following either overexpression of the SP4 isoform or down regulation using shRNA.

SP4 cDNA (n = 5) and shRNAs (n = 4) were transduced via adenoviral delivery. p<0.0001 by ANOVA. *p<0.05 compared to luciferase control shRNA, ** p<0.01 compared to luciferase control shRNA (n = 5) using log transformed data.

Figure 2. CADM1 is responsible for mast cell adhesion to airway smooth muscle cells.

HMC-1 cells and HLMCs were transduced with SP4, SP1, CADM1 shRNA, GFP or luciferase control shRNA using adenoviral delivery, followed by the study of adhesion to HASMCs. A) Downregulation of CADM1 significantly reduced HMC-1 cell adhesion to HASMCs (n = 4), but overexpression of the SP4 or SP1 isoforms did not increase it. *p<0.05, **p<0.01 compared to luciferase control shRNA. B) Downregulation of CADM1 using shRNA reduced adhesion of HLMCs (n = 4) to HASMCs (n = 3, except for luciferase control shRNA where n = 2), but overexpression of SP4 or SP1 did not increase it. *p<0.05 compared to CADM1 shRNA.

Figure 3. CADM1 is responsible for mast cell adhesion to primary human lung fibroblasts.

HMC-1 cells and HLMCs were transduced with SP4, SP1, CADM1 shRNA, GFP or luciferase control shRNA using adenoviral delivery, followed by the study of adhesion to HLFs. A) Downregulation of CADM1 reduced HMC-1 cell adhesion to HLFs (n = 3–6 for different groups), while overexpression of SP4 increased it. *p<0.05, **p<0.01 compared to CADM1 shRNA. #p<0.05 compared to SP4. B) Downregulation of CADM1 reduced adhesion of HLMCs (n = 4) to HLFs (n = 5), while SP4 overexpression increased adhesion. *p<0.05 compared to CADM1 shRNA. #p<0.05 compared to SP4. C) HMC-1 cells pre-incubated with anti-CADM1 9D2 Ab adhered less to HLFs compared to controls (n = 3). **p<0.01 compared to 9D2. D) and E) Scatter plots with regression model parameters for the CADM1 surface expression levels and the degree of adhesion to HLFs by HMC-1 cells (D) and HLMCs (E), which include the data for transduced cells from Fig. 3 including SP4, luciferase control shRNA and CADM1 shRNA transduction groups (20 points in D and 14 points in E) and their expression data published previously in Fig 2b of reference 26 for HMC-1 cells and Fig. 1 in this paper for HLMCs.

Figure 4. CADM1 counter-receptors in parenchymal human lung fibroblasts and airway smooth muscle cells.

A) Western immunoblot of proteins (25 µg/lane) from HLFs and HASMCs from 3 donors each or HMC-1 cells for CADM1 counter-receptors (shown on the right) with molecular markers (shown on the left). B) Quantification of CADM1, nectin-3 and β-actin levels in HLFs and HASMCs relative to HMC-1 cells. *p<0.05, **p<0.01 compared to ASMCs.

Figure 5. CADM1 promotes lung fibroblast-dependent HLMC survival and proliferation.

HLMC numbers (n = 3 donors) after 5 days culture in media alone, or co-culture with HLFs (n = 3 donors). CADM1-adhesion blocking antibody or isotype control were added where indicated. 12500 HLMCs were present at initiation of the cultures. *p<0.05, ** p<0.01. Statistics performed on log transformed data, zero values for cell counts given nominal value of 100 for log transformation.