Comparative Ability of Mesenchymal Stromal Cells from Different Tissues to Limit Neutrophil Recruitment to Inflamed Endothelium

Abstract

Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton's jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary.

Conflict of interest statement

Competing Interests: HMM has received research funding from Pfizer. This does not alter the authors' adherence to PLoS ONE policies on sharing data and materials. All other authors declare that they have no conflicts of interest.

Figures

Fig 1. Characterisation of MSC isolated from different tissue sources.

(A) Expression of MSC markers CD44, CD73, CD90, CD105, CD146, CD271 and combined expression of the haematopoietic markers CD14, CD20, CD34 and CD45 by BMMSC, WJMSC, or TBMSC by flow cytometry. Data are expressed as representative histograms compared to isotype controls and are representative of n = 3 independent experiments using 3 different MSC donors. (B) BMMSC, WJMSC, and TBMSC were differentiated towards osteoblast, adipocyte, or chrondocyte lineage for 14 or 21 days. Adipogenic, osteogenic and chrondogenic differentiation were assessed using Oil Red O, Alizarin Red or Alcian Blue to stain lipid droplets, calcium deposits or cartilage specific proteoglycans respectively. Phase contrast and colour micrographs are representative of n = 3 independent experiments using 3 different MSC donors. Scale bar represents 10μm.

Fig 2. Effect of MSC from different tissues on neutrophil recruitment when cultured in direct contact with EC.

BMMSC, WJMSC, or TBMSC were cultured in direct contact with EC in channel slides for 24h before addition of 100U/ml TNFα for 4h. (A) MSC labelled with Cell Tracker Green integrated into the EC monolayer. Fluorescent micrographs are representative of n = 3 independent experiments using 3 different MSC donors. (B) Ratio of MSC to EC for each co-culture assessed after the adhesion assay. ANOVA showed a significant effect of MSC source on MSC:EC ratio, p

Fig 3. Effect of MSC from different tissues on neutrophil recruitment when cultured on the opposite side of the filter to EC.

MSC and EC were cultured on opposite sides of 0.4μm porous filters for 24h prior to stimulation with 100U/ml TNFα for 4h. Co-cultures formed with (A-B) MSC from different sources (BM, WJ, TB) or (C-D) with different numbers of WJMSC. Neutrophil (A,C) adhesion and (B,D) migration were expressed as a proportion of values observed on the paired EC mono-culture control. In A and C, ANOVA showed a significant effect of culture conditions on neutrophil adhesion, p5 cells where 3 different donors were used * = p<0.05 and ** = p<0.01 compared to EC mono-cultures by Dunnett post-test.

Fig 4. Effects of passage on the ability of MSC to suppress neutrophil recruitment.

(A) BMMSC or (B) WJMSC at different passage number were co-cultured with EC on opposite sides of a porous filter for 24h prior to stimulation with TNFα for 4h. Neutrophil adhesion was expressed as a proportion of that observed on the paired EC mono-culture control. In A and B, ANOVA showed a significant effect of passage on neutrophil adhesion, p

Fig 5. Effects of varying proximity between MSC and EC on neutrophil recruitment.

Co-cultures were formed by seeding BMMSC, WJMSC, or TBMSC on the opposite side of a Transwell filter to EC (Close proximity) or by seeding MSC on the plate below EC cultured on a filter (Separate). Neutrophil adhesion was expressed as a proportion of that observed on the paired EC mono-culture control where a different EC and neutrophil donor was used in each experiment. ANOVA showed a significant effect of culture conditions on neutrophil adhesion for each type of MSC, p

Fig 6. Effects of media conditioned by MSC alone or in co-culture with EC on neutrophil recruitment.

EC mono-cultures were treated with conditioned media from BMMSC, WJMSC, or TBMSC either cultured alone or co-cultured with EC on opposite sides of filters for 24h. Not all conditions were performed in all experiments, but treated cells were always compared to paired untreated EC controls. Neutrophil adhesion was assessed at 2min post-perfusion and expressed as a proportion of that observed on the paired EC mono-culture control. ANOVA showed a significant effect of culture conditions on neutrophil adhesion for each type of MSC, p

Fig 7. Effect of blocking the actions of IL-6 on the immunosuppressive effects of MSC in co-culture.

(A) BMMSC or (B) WJMSC co-cultures were cultured in close proximity, on opposite sides of a porous filters for 24h and then treated with neutralising antibodies against IL-6 or a function blocking antibody against IL-6R for the duration of the co-culture and cytokine treatment. Neutrophil adhesion was expressed as a proportion of that observed on the paired EC mono-culture. ANOVA showed a significant effect of co-culture treatment on neutrophil adhesion in (A) (p

Fig 8. Effects of co-culture, of proximity between MSC and EC and MSC passage on IL-6 secretion.

MSC-EC co-cultures were formed (A) in direct contact using channel slides; (B, D-E) in close proximity on opposite sides of a porous filter; or (C) in close proximity or with MSC seeded below and separate from EC on the filter above. EC and MSC mono-cultures (at p3 for WJMSC and p5 for BMMSC) were set up as controls. IL-6 release into supernatants was assessed after 24h. In (D) and (E), ANOVA showed a significant effect of culture conditions, p