Mechanisms of adhesion and subsequent actions of a haematopoietic stem cell line, HPC-7, in the injured murine intestinal microcirculation in vivo

Abstract

Objectives: Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment.

Methods: Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1β or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally.

Results: IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12.

Conclusion: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential.

Figure 1. HPC-7 adhesion in vitro and in vivo is increased on IR injured intestine.

(A) HPC-7 adhesion in vitro was raised on frozen ileal sections isolated from IR injured animals when compared to sham controls. Results represent mean adhesion per 1×10⁴ µm² tissue area±SEM (n>4/group); * p<0.05. (B) HPC-7 adhesion in ileal mucosal microcirculation in vivo was also raised in animals subjected to IR injury (solid line: sham; dashed line: IR injury). (C) Free-flowing HPC-7 cells were increased in IR mice (solid line: sham; dashed line: IR injury). (D) IR injury enhanced HPC-7 adhesion within jejunal villi when quantitated ex vivo. (E) IR injury did not enhance HPC-7 adhesion to duodenum when quantitated ex vivo. Representative images of the villous microcirculation of the ileum in (F) sham and (G) IR animals are shown. Results are presented as mean adhesion±SEM (n≥7/group); * p<0.05. For frozen section work, data is expressed as mean adhesion per 1×10⁴ µm² of tissue to control for area variation.

Figure 2. Recruited HPC-7 do not reduce leukocyte infiltration in IR injured intestine.

(A) Leukocyte infiltration, analysed by AcrO staining, did not reduce in animals receiving 2×10⁶ HPC-7 cells at 30 minutes post-reperfusion when compared to IR injured animals receiving a saline bolus ie. no cells. Representive images from the ileum of IR treated animals receiving (B) saline or (C) 2×10⁶ HPC-7 are shown. Results are presented as mean cells per field±SEM.

Figure 3. HPC-7 and KSL cells express CD18 and CD49d.

HPC-7 cells express (A) CD11a, but not (B) CD11b or (C) CD11c. Flow cytometry also revealed expression of (D) CD18 and (E) CD49d on the surface of HPC-7. (F) Whole bone marrow was depleted for lineage postive cells and labelled for Sca-1, c-kit and either IgG, CD18 or CD49d. The gating used is shown in panel F (FL2: Sca-1; FL3: c-Kit). (G) KSL cells express cell surface CD49d, while CD18 could not be detected. (H) CD18 and CD49d could be identified by flow cytometry following cell permeabilization.

Figure 4. HPC-7 adhesion in vivo to intestinal microcirculation is dependent on CD18.

(A) Pre-treatment of HPC-7 with an anti-CD18 antibody reduced adhesion in the IR injured gut microcirculation (solid line: IgG; dashed line: anti-CD18). (B) Pre-treatment with an anti-CD18 antibody did not significantly reduce free-flowing cells in the ileum (solid line: IgG; dashed line: anti-CD49d). (C) Pre-treatment of HPC-7 with anti-CD49d antibody did not reduce their adhesion in the IR injured gut microcirculation (solid line: IgG; dashed line: anti-CD49d). (D) Pre-treatment of HPC-7 with anti-CD49d antibody significantly reduced free flowing cells in the ileum (solid line: IgG; dashed line: anti-CD49d). (E) Treatment of HPC-7 with an anti-CD18 antibody reduced adhesion in the jejunum. Results are presented as mean adhesion per field±SEM (n≥4/group); * p<0.05.

Figure 5. HPC-7 adhesion in vivo is increased in IR injured muscle and is dependent on CD49d.

(A) HPC-7 adhesion to cremaster PCVs was enhanced following IR injury. (B) Pre-treating HPC-7 with an anti-CD18 or anti-CD49d blocking antibody reduced HPC-7 adhesion. (C) IR injury enhanced HPC-7 rolling in PCVs. (D) Blocking with anti-CD18 or anti-CD49d antibodies did not enhance rolling above that seen with IR injury alone. (E) Representative images (x10 magnification) of HPC-7 adhesion in sham and IR injury are shown. Results are presented as mean adhesion±SEM (n≥3/group); A/C: * p<0.05, IR vs Sham; B: * p<0.05, ** p<0.01, IR vs antibody blocked cells.

Figure 6. Topical treatment of the small intestine with cytokines increases HPC-7 adhesion in vivo.

(A) Mucosal treatment with TNF-α significantly enhanced adhesion of HPC-7 cells in vivo. (B) No increase in free-flowing HPC-7 was observed following topical treatment of the gut with TNF-α. (C) Mucosal treatment with IL-1β significantly enhanced adhesion of HPC-7 cells in vivo. (D) No increase in freeflowing HPC-7 was observed following topical treatment of the gut with IL-1β. (E) No increase in adhesion was observed following topical treatment of the gut with CXCL12. (F) Significantly increased numbers of HPC-7 were observed free-flowing through the gut following topical CXCL12 treatment. Results are represented as mean cells adherent/field±SEM (n≥3/group); * p<0.01, ** p<0.01, PBS topical treatment vs Cytokine topical treatment.

Figure 7. TNF-α and IL-1β significantly enhance adhesion in the cremaster but this is not augmented with topical treatment of the cremaster with CXCL12.

Cremasteric exposure to only TNF-α and IL-1β promoted HPC-7 adhesion when monitored in (A) individual PCVs or (B) the entire cremaster. This adhesion was not increased further in the presence of CXCL12. (C) Rolling was affected by IL-1β and CXCL12 alone and was enhanced further in the presence of both together. Results are presented as mean cells adherent per PCV, mean rolling cells per PCV and mean cells adherent per cremaster±SEM (n = 3/group); * p<0.05, ** p<0.01, *** p<0.001 (vs vehicle with no CXCL12); #p<0.05 (vs TNF-α with no CXCL12); ++p<0.01 (vs vehicle with CXCL12); ▪▪ p<0.01 (vs IL-1β with no CXCL12).

Figure 8. HPC-7 pre-treatment with CXCL12 enhances their adhesion on colonic endothelium in vitro, IR injured intestine in vivo and reduces leukocyte infiltration post-IR injury.

(A) HPC-7 express the cytokine receptors TNFR1, TNFR2, IL-1RI and CXCR4. (B) Pre-treatment of HPC-7 with 100 ng/ml IL-1β or 100 ng/ml CXCL12 enhances adhesion of HPC-7 on TNF-α activated colonic endothelium in vitro. Pre-treatment of HPC-7 with (C) 100 ng/ml TNF-α or (D) 100 ng/ml IL-1β does not enhance intestinal HPC-7 adhesion following IR injury in vivo. (E) Pre-treatment of HPC-7 with 100 ng/ml CXCL12 enhances adhesion in the intestine following IR injury in vivo. (F) Leukocyte infiltration, analysed by AcrO staining, was reduced at four hours in animals receiving 2×10⁶ CXCL12 pre-treated HPC-7 cells at 30 minutes post-reperfusion when compared to IR animals receiving a saline bolus. Results are presented as mean adherent cells per field±SEM (Figure B, n = 3; Figures C-E, n≥3; Figure F, n≥12); * p<0.05, ** p<0.01, *** p<0.001 vs control).

Figure 9. Pre-treating HPC-7 with CXCL12 increases their binding to endothelial counterligands VCAM-1 and ICAM-1, but does not increase levels of surface integrin expression.

No up-regulation of (A) CD18 or (B) CD49d is identifiable by flow cytometry following treatment of HPC-7 with CXCL12, TNF-α or IL-1β. Pre-treatment of HPC-7 with CXCL12 enhances HPC-7 adhesion on (C) ICAM-1 and (D) VCAM-1. Treating HPC-7 with CXCL12 for an hour significantly enhances the (E, top left) number, but not size (E, bottom left) of membrane CD18 clusters when compared to PBS controls. Conversely, treating HPC-7 with CXCL12 for an hour significantly enhances the size (E, bottom right) but not number (E, top right) of CD49d clusters on the surface of HPC-7. Representative images are shown in (F). Results are presented as representative flow cytometry plots (Figure A, B, representative plot from n = 3), mean adherent HPC-7/per field, normalized to adhesion on a BSA control±SEM (Figure C and D, n = 3/group) and cluster count/size as analysed by ImageJ (Figure E, n = 3/group); * p<0.05 (vs vehicle treated).