The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo

Abstract

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Figure 1. Development of a 4D imaging platform for analysis of leukocyte TEM in vivo

Cremasteric venules of Lys-EGFP-ki mice (exhibiting green leukocytes) were immunostained in vivo for EC junctions with intrascrotal (i.s.) injection of Alexa Fluor-555-labeled anti-PECAM-1 mAb 390 (PECAM-555; 3μg/mouse; shown in red), and were stimulated with i.s. IL-1β (50 ng/mouse). 2 h later tissues were surgically exteriorised and images were captured at 40x magnification by in vivo confocal intravital microscopy (IVM) at 1min intervals for a period of 90min. (a) Panels show images of a post-capillary venular segment immediately after tissue exteriorisation (at t=120, 160, and 210 min post IL-1β application), showing the development of an inflammatory response (Supplementary Video 1). (b) and (c) show that intravenous (i.v.) or i.s. mAb 390 has no effect on leukocyte adhesion or transmigration induced by IL-1β as analyzed by brightfield IVM. (b) WT mice were pretreated with i.v. mAb 390, Mec13.3 (a blocking anti-PECAM-1 mAb), an IgG2b isotype control mAb (all at 3mg/kg) prior to i.s. administration of IL-1β, or were untreated. 4 h later, tissues were exteriorized and leukocyte responses of adhesion and transmigration were quantified by IVM. (c) WT mice were injected with i.s. saline or IL-1β, with or without i.s. Alexa Fluor-555-labeled mAb 390 (3μg). After 4 h leukocyte adhesion and transmigration responses were quantified by IVM (n = 3-8 animals, 3-5 vessels per animal. Error bars show S.E.M). Statistically significant responses in IL-1β-stimulated tissues as compared to saline-injected cremaster muscles are indicated by asterisks, *P<0.05 and **P<0.01. Other statistical comparisons are shown by ***P<0.001.

Figure 2. Analysis of neutrophil paracellular and transcellular TEM in vivo

TEM responses were imaged in IL-1β-stimulated PECAM-1-labelled (red) tissues of Lys-EGFP-ki mice (leukocytes shown in green). (a) Paracellular TEM response of a leukocyte (*, upper panels) and its associated transient junctional pore formation (lower panels) (Supplementary Video 3). (b) Transcellular TEM event illustrating migration through ECs with no disruption of PECAM-1 enriched junctions. False color images of the PECAM-1 channel (high and low intensity sites shown in white and blue, respectively) enable visualization of the transcellular pore (arrow) (Supplementary Video 4). (c) Additional examples of paracellular TEM and (d) transcellular TEM events. Some transcellular pores occurred in close proximity to EC junctions without disrupting PECAM-1-labelled junctions (right panel). (e-f) Linear intensity profiles of PECAM-555 (EC; red) and GFP (leukocyte; green) of TEM events along the indicated dotted lines shown in (c) and (d). (e) The paracellular pore is immediately flanked by high PECAM-1-labelled junctions (Jn), while in (f) the transcellular pore is formed in the EC body where lower PECAM-1 labeling is observed. Intensity profiles post TEM illustrates pore closures. Scale bars = 10μm. The frequency (g) and duration (h) of TEM responses as induced by multiple stimuli was quantified (n > 103 TEM events observed in 4-7 mice; mean ± S.E.M). Statistical significance between frequency of non-junctional and bi-cellular TEM events (*P < 0.01 and ** P < 0.001) and between non junctional and multi cellular locations (***P < 0.05 and †P < 0.001) are indicated.

Figure 3. Observation and quantification of disrupted forms of polarized paracellular TEM

Disrupted polarised paracellular TEM behaviours, termed “hesitant” and “reverse” TEM were observed. (a) Time-lapse images of a GFP-leukocyte (*) exhibiting hesitant paracellular TEM. Top panels show a transverse section of the venule, middle panels show the event from the luminal side as supported by illustrative drawings (lower panels), showing the sub-EC segments of the migrating leukocyte in light green with dashed outline (Supplementary Video 5). Additional examples are shown in Supplementary Figure 2 and Supplementary Videos 6 and 7. (b) Time-lapse images of a leukocyte exhibiting reverse TEM. The leukocyte migrates through a bi-cellular junction in an abluminal to luminal direction, disengages from the junction and crawls away on the luminal surface. Top panels show transverse sections of the venule and bottom panels show the event from the luminal side (Supplementary Video 8). (c) Frequency of normal, hesitant and reverse paracellular TEM as induced by multiple stimuli expressed as a % of total paracellular TEM events (n ≤ 103 TEM events as observed in 4-7 mice per inflammatory reaction). (d) The duration of normal, hesitant and reverse TEM events in I-R injured tissues were analyzed. Statistical differences between normal and disrupted (hesitant and reverse) TEM events (*P< 0.001), and differences between hesitant and reverse TEM events (**P< 0.001) are indicated. Data are shown as mean ± S.E.M.

Figure 4. Neutrophils exhibit disrupted forms of polarized paracellular TEM

A number of approaches demonstrated that within the employed models the leukocyte sub-type that exhibited disrupted forms of polarized TEM were neutrophils. (a) I-R-induced TEM was analyzed in control and neutrophil depleted Lys-EGFP-ki mice (Supplementary Figure 4a). The frequency of normal and disrupted TEM events (both reverse and hesitant) per 30 min (standard image sequence capture time) is shown (results are from n=7-9 animals). (b) The GFP intensity of monocytes (CD115⁺; n=81) and neutrophils (CD115⁻; n=158) (Supplementary Figure 4b) in CCL2-stimulated Lys-EGFP-ki mouse cremaster muscles (n = 4 animals) was quantified from 2D projections at standard (routinely employed and optimized for analysis of GFP⁺ neutrophils) and high GFP gain image capture settings, and compared to the threshold intensity for visibility in 3D reconstructions (indicated by a dotted line; intensity of ~200 Grays/μm²). (c) A 3D reconstruction image of a CCL2-stimulated Lys-EGFP-ki mouse cremasteric venule, with anti-PECAM-1-labeled EC junctions (red), acquired using standard GFP gain settings. The image shows the visibility of GFP⁺ neutrophils (green) but not monocytes, immunostained with i.v. anti-CD115 mAb (blue), using the standard GFP settings. Statistically significant differences between control and neutrophil depleted mice (*P<0.05 and **P<0.01), or between background and leukocyte-associated fluorescence intensity (**P<0.01 and ***P<0.001) are shown. Data are shown as mean ± S.E.M. Scale bars = 10 μm.

Figure 5. JAM-C is disrupted from EC junctions in response to I-R injury but not IL-1β

The expression and localisation of JAM-C was investigated on post capillary cremasteric venules in un-stimulated tissues, IL-1β-stimulated tissues and tissues subjected to I-R injury by immunofluorescence staining and immunoelectron microscopy. One group of mice was also pretreated with superoxide dismutase (SOD) and catalase (2,000 and 50,000 U/kg respectively, i.v.) prior to induction of I-R injury. (a) Cremasteric venules of WT mice were immunostained ex-vivo for VE-Cadherin (green), PECAM-1 (red) and JAM-C (blue; a JAM-C⁺ nerve [N] is also shown). Images are representative from n = 7-15 mice/treatment group, 4-10 vessels per mouse. Scale bars=10 μm. The fluorescence intensity at EC junctions was quantified (see Supplementary Methods) and presented in (b). Statistically significant differences in protein intensity between stimulus and treatment groups were analysed by ANOVA and are indicated by asterisks *P<0.05 and **P<0.01. Data are shown as mean ± S.E.M. (c) The distribution of JAM-C on ECs was also analyzed by immunoelectron microscopy in control tissues (saline-injected or sham-operated) and cremaster muscles stimulated with IL-1β or I-R injury. Examples of images acquired are shown in Supplementary Fig. 5a and b. Quantification revealed that JAM-C was significantly displaced from junctional regions in I-R, but not IL-1β-stimulated tissues (17-33 ECs from n=2-5 mice/group were quantified and analyzed by Chi-square test, ***P<0.001).

Figure 6. EC JAM-C plays a critical role in mediating polarized neutrophil paracellular TEM

The functional role of EC JAM-C in mediating disrupted polarized forms of paracellular TEM was investigated through pharmacological blockade and genetic reduction of EC JAM-C. (a) Lys-EGFP-ki mice were subjected to I-R injury and different groups of mice were pretreated with i.v. saline, a control non-blocking anti-JAM-C mAb (H36) or with a blocking anti-JAM-C mAb (H33) (mAbs given at 3mg/kg). The occurrence of normal, hesitant and reverse TEM responses was quantified and presented as a % of total paracellular TEM events. Statistically significant differences in data sets, as analyzed by multinomial logistic regression analysis, are shown by asterisks (*P<0.05 and **P<0.01). (b) The occurrence of all disrupted TEM events (hesitant and reverse) expressed as a % of total paracellular responses as induced by I-R injury in control Lys-EGFP-ki mice and Lys-EGFP-ki mice pretreated with anti-JAM-A mAb, anti-JAM-C mAb or soluble JAM-C ( all given i.v. at 3 mg/kg) and Lys-EGFP-ki mice crossed with Tie2Cre:JAM-C^flox/flox mice (exhibiting EC JAM-C reduction and statistically compared with littermate controls exhibiting normal EC JAM-C) are shown. These results are compared with % of disrupted TEM events in IL-1β-stimulated tissues in un-treated and anti-JAM-C mAb pretreated Lys-EGFP-ki mice and in Lys-EGFP-ki mice crossed with Tie2Cre:JAM-C^flox/flox mice. Results are from 43-109 TEM events as quantified in n = 4-10 mice. Data are shown as mean ± S.E.M. Statistically significant differences as analysed by ANOVA, between control and experimental groups are indicated by asterisks (*P<0.05).

Figure 7. Reverse transmigrated neutrophils are associated with pulmonary inflammation following I-R injury

(a) Reverse transmigrated (rTEM) bone-marrow (BM) murine neutrophils (PMN) were generated in vitro (Supplementary Methods) and ICAM-1 expression on rTEM and fresh PMN as well as multiple other PMN preparations was analysed by flow cytometry (right panel; n≥3 preparations). (b-g) Cremasteric and lower limb I-R procedures were performed. Post I-R, mice were exsanguinated and pulmonary vascular washout leukocytes collected. (b) Lung tissue neutrophil infiltrate was quantified by flow cytometry of digested and homogenised tissues. (c) Oedema formation was quantified by local accumulation of i.v. injected Evans blue. (d) Pulmonary vascular washout neutrophils from sham (left) and limb I-R (right) animals were analysed for ICAM-1 expression by flow cytometry and examples of scatter plots of ICAM-1 and FSC profiles are shown. The numbers in the gates demonstrate the greater % of ICAM-1^hi/FSC^hi population in the I-R group. (e) Pulmonary vascular washout ICAM-1^lo and ICAM-1^hi neutrophils were analysed for ROS generation (both panels) with lymphocytes acting as a negative control (right). (f) The total number of ICAM-1^hi neutrophils in the pulmonary vascular washout of mice subjected to sham and I-R protocols was analysed. (g) Collation of all I-R stimulated groups shows a positive correlation between the % of ICAM-1^hi pulmonary vascular and the number of tissue infiltrated neutrophils. Data are shown as mean ± S.E.M (n= 6–9 animals per group). Statistical differences between control and experimental groups, and correlations, are shown with asterisks (*P<0.05, **P<0.01, ***P<0.001). Additional comparisons are indicated by lines (†P<0.001).