IL-1β-Mediated Activation of Adipose-Derived Mesenchymal Stromal Cells Results in PMN Reallocation and Enhanced Phagocytosis: A Possible Mechanism for the Reduction of Osteoarthritis Pathology

Abstract

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1β levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1β or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASC_NS-CM) or IL-1β-stimulated ASCs (ASC_IL-1β-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1β-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1β-stimulated ASCs. Also migration of PMNs toward ASC_IL-1β-CM was significantly enhanced (287%) when compared to ASC_NS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASC_IL-1β-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

Keywords: CiOA; PMNs; adipose-derived mesenchymal stromal cells; chemokines; interleukin-1β; phagocytosis; synovitis.

Figure 1

PMNs reallocate and cluster with ASCs in knees with early CiOA after intra-articular ASC injection. (A,B) Intra-articular injection on day 7 of CiOA resulted in attraction of immune cells within 6 h, both in the control (A) and ASC-injected (B) joints, as was shown in HE-stained total knee joint sections. (C,D) Higher magnifications showed that the immune cells in both saline- (C) and ASC-injected (D) CiOA knee joints had a polymorphonuclear (PMN) phenotype. (E,F) Immunohistochemistry with the specific antibody NIMP-R14 confirmed that a large number of PMNs is attracted to the joints. They were equally spread throughout the synovium and the joint cavity in the control-injected joints (E), but in the ASC-injected joints a remarkable accumulation of PMNs along the lining was found (F) as indicated by arrows. (G) NIMP-R14 staining showed that 24 h after ASC injection the PMN influx had largely disappeared. (H) ASC injection in naïve joints resulted in NIMP-R14 positive cell influx, albeit lower than in CiOA joints. (I) Quantification of attracted PMNs after intra-articular injection confirmed a significantly increased number of PMNs in CiOA joints compared to naïve joints. (J) At higher magnification we observed a clustering of PMNs around cells we supposed to be ASCs (arrow), which was confirmed with a CD271 antibody (K) which specifically stains ASCs (arrows). Control joints were CiOA knees injected with saline supplemented with 1% BSA. Images shown are representative for the treatment groups (n = 8 per group). Original magnification × 200 (A,B,E,F,G,H) or × 1,000 (C,D,J,K). Differences between groups were tested using a one-way ANOVA followed by a Bonferroni Multiple Comparison posttest. F, femur; S, synovium. Bars show mean values ± SD. *P < 0.05.

Figure 2

Expression of PMN-attracting chemokines by ASCs is elevated by IL-1β in vitro. The effect of an inflammatory milieu on gene expression in ASCs was assessed by 24 h stimulation with different pro-inflammatory mediators. (A) Murine ASCs showed a significantly increased gene expression of PMN-attracting chemokines CXCL5, CXCL7, and KC after stimulation with IL-1β. (B) Also on protein level in the supernatant, KC was significantly up-regulated after IL-1β stimulation. Human ASCs show a comparable elevated level of the functional KC-homolog IL-8 after IL-1β stimulation on both gene expression (C) and protein level (D). Gene expression levels are presented as –ΔC_t compared to GAPDH. Closed circles represent murine samples, open triangles represent human samples [n = 4 (A,B) or 3 (C,D) per group]. Differences between non-stimulated (NS) ASCs and the several stimulations were tested using a one-way ANOVA followed by a Dunnett's posttest. Bars show mean values ± SD. ***P < 0.001 vs. NS ASCs.

Figure 3

Clustering of murine PMNs with ASCs is enhanced by IL-1β in vitro. The association of freshly isolated murine PMNs with adherent ASCs driven by IL-1β was assessed in a co-culture experiment. (A) After 6 h, clustering cells were stained with May-Grünwald Giemsa and quantified. Large cells are ASCs, small cells are PMNs. Both the number of ASCs that formed clusters with PMNs (B) and the average number of PMNs clustered per ASC (C) were significantly increased in presence of IL-1β when compared to the non-stimulated co-cultures (NS). N = 3 per group, quantified at five separate areas per sample. Differences between the groups were tested using a Student's t-test. Bars show mean values ± SD. *P < 0.05, **P < 0.01.

Figure 4

PMN attraction by ASCs is increased by IL-1β in vitro. The potential of IL-1β-stimulated ASCs to attract PMNs was investigated using transwell inserts. (A) KC levels in conditioned medium (CM) of IL-1β-stimulated ASCs (ASC_IL-1β-CM, no exogenous IL-1β present) were significantly elevated compared to CM of non-stimulated ASCs (ASC_NS-CM). (B) Migration of freshly isolated PMNs toward ASC_IL-1β-CM as well as toward the positive control KC was significantly increased compared to ASC_NS-CM. No enhanced migration of PMNs was found in the negative controls containing MCP-1 or IL-1β. The number of migrated PMNs was expressed as percentage of total cells added to the top compartment [n = 3 (A) or 6 (B) per group]. Differences between groups were tested using a Student's t-test (A) or a one-way ANOVA followed by a Dunnett's posttest (B). Bars show mean values ± SD. ***P < 0.001 vs. ASC_NS-CM.

Figure 5

IL-1β-mediated release of inflammatory factors is significantly lowered after clustering of PMNs with ASCs. The effect of clustering of PMNs with ASCs on protein release was determined. (A) KC release by ASCs was already significantly increased after 3 and 6 h of co-culture with PMNs in presence of IL-1β. After 24 and 48 h of co-culture with IL-1β, clustering of PMNs with ASCs resulted in a significantly lowered KC release when compared to ASCs cultured alone. (B) S100A8/A9 release was not detectable in a monoculture of ASCs, but PMNs released basal levels of S100A8/A9. These levels increased over time, but remained lower in co-cultures of PMNs with ASCs in presence of IL-1β. N = 3 per group. Differences between groups were tested using a one-way ANOVA followed by a Bonferroni Multiple Comparison posttest for each time point separately. Bars show mean values ± SD. ***P < 0.001. ND, not detectable.

Figure 6

Phagocytosis by PMNs is enhanced after priming with conditioned medium of IL-1β-stimulated ASCs. The immunomodulatory effect of ASCs on PMNs was investigated by assessment of the phagocytic capacity after priming with ASC-conditioned medium. (A) The uptake of labeled zymosan particles by murine PMNs primed with conditioned medium (CM) of non-stimulated ASCs (ASC_NS-CM) was significantly enhanced when compared to the negative control containing fresh culture medium. Incubation of PMNs with CM of ASCs which were stimulated with IL-1β (ASC_IL-1β-CM, no exogenous IL-1β is present) further enhanced phagocytosis when compared to ASC_NS-CM. (B) Human PMNs primed with ASC_NS-CM showed a similar increase in phagocytic capacity when compared to culture medium, which was even further enhanced after priming with ASC_IL-1β-CM. Closed circles represent murine samples, open triangles represent human samples (n = 3 per group). Differences between groups were tested using a one-way ANOVA followed by a Bonferroni Multiple Comparison posttest. Bars show mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.