Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling

Abstract

Two key characteristics of the inflammatory response are the recruitment of leukocytes to inflamed tissue as well as changes in vessel permeability. We explored the relationship between these two processes using intravital confocal microscopy in cremasters of anesthetized (65 mg/kg Nembutal ip) mice. We provide direct evidence that intercellular adhesion molecule-1 (ICAM-1) links leukocyte-endothelial cell interactions and changes in solute permeability (Ps). Importantly, we show that arterioles, not just venules, respond to proinflammatory stimuli, thus contributing to microvascular exchange. We identified two independent, ICAM-1-mediated pathways regulating Ps. Under control conditions in wild-type (WT) mice, there is a constitutive PKC-dependent pathway (Ps = 1.0 +/- 0.10 and 2.2 +/- 0.46 x 10(-6) cm/s in arterioles and venules, respectively), which was significantly reduced in ICAM-1 knockout (KO) mice (Ps = 0.54 +/- 0.07 and 0.77 +/- 0.11 x 10(-6) cm/s). The PKC inhibitor bisindolylmaleimid l (1 micromol/l in 0.01% DMSO) decreased P(s) in WT mice to levels similar to those in ICAM-1 KO mice. Likewise, a PKC activator (phorbol-12-myristate-acetate; 1 micromol/l in 0.01% DMSO) successfully restored Ps in ICAM-1 KO vessels to be not different from that of the WT controls. On the other hand, during TNF-alpha-induced inflammation, Ps in WT mice was significantly increased (2-fold in venules and 2.5-fold in arterioles) in a Src-dependent and PKC-independent manner. The blockade of Src (PP2; 2 micromol/l in 0.01% DMSO) but not PKC significantly reduced the TNF-alpha-dependent increase in Ps. We conclude that ICAM-1 plays an essential role in the regulation of Ps in microvessels and that there are two separate (constitutive and inducible) signaling pathways that regulate permeability under normal and inflamed conditions.

Fig. 1.

TNF-α-mediated increase in solute permeability (P_s) is ICAM-1 dependent. A: P_s in wild-type (WT) arterioles and venules significantly increased following TNF-α treatment (n = 17 vessels). B: in ICAM-1 knockout (KO) mice, TNF-α-induced increase in P_s was abolished in arterioles and venules (n = 12). C: overlay of average P_s values from WT and ICAM-1 KO mice. ICAM-1 KO vessels were much less permeable than WT vessels, and the difference between arteriolar and venular P_s was abolished. *Significantly different from controls and each other; +significantly different from each other (P < 0.05).

Fig. 2.

β₂-integrins (CD18) expression is not different in WT and ICAM-1 KO mice. To measure total CD18 expression on mouse leukocytes, flow cytometry was performed on white cells from whole blood sample, which were labeled with rat anti-mouse FITC-conjugated anti-CD18 monoclonal antibody (clone M18/2). Corresponding isotype FITC-conjugated rat IgG2a (clone YTH71.3) was used as a negative control. n = 5,000 cells for each sample. A: WT. B: ICAM-1 KO. Solid line, CD18-positive cells; shaded areas, isotype control. C: overlay of CD18-positive WT and ICAM-1 KO mice. Solid line, KO; shaded area, WT.

Fig. 3.

ICAM-1 ligation increased P_s in arterioles but not in venules. In control WT arterioles, but not WT venules, antibody (Ab) ligation of ICAM-1 (YN-1/1.7.4; 100 μg iv) significantly increased P_s to levels similar to those following TNF-α activation (P < 0.05; n = 10 vessels). x is significantly different from WT without ICAM-1 ligation (Fig. 1A).

Fig. 4.

CD18/ICAM-1 interactions are essential for TNF-α-mediated P_s increase. A: P_s in control and TNF-α-activated microvessels in CD18 KO mice. P_s in control vessels was similar to that in WT, but P_s did not increase with TNF-α. B: CD18 blocking antibody (CD18 GAME-46; 100 μg iv), but not IgG control (R3-34; 100 μg iv), significantly reduced the TNF-α-induced increase in P_s in WT arterioles and venules. x is significantly different from P_s measured in TNF-α-treated WT mice (n = 10 vessels; Fig. 1A); *significantly different (P < 0.05) from controls (n = 9 vessels).

Fig. 5.

P_s under control conditions is PKC dependent. A: control WT vessels treated with DMSO alone, bisindolylmaleimid l (BIM; PKC blocker in 0.01% DMSO, 1 μmol/l for 10 min in superfusate), and ICAM-1 KO mice with DMSO alone. DMSO had no effect in both WT and ICAM-1 KO (P_s similar to Fig. 1, A and B). P_s significantly decreased with BIM to levels similar to ICAM-1 KO mice. B: control ICAM-1 KO and WT vessels treated with PMA (PKC activator in 0.01% DMSO, 1 μM for 10 min in superfusate). P_s in ICAM-1 KO vessels was restored to WT levels, whereas P_s in WT vessels was unchanged. * and ^, Significantly different (P < 0.05) from each other (n = 10 vessels); x is significantly different (P < 0.05) from P_s measured in ICAM-1 KO mice (n = 10 vessels; Fig. 1B).

Fig. 6.

TNF-α-mediated increase in P_s is Src dependent. A: control WT vessels treated with TNF-α or TNF-α + BIM (in 0.01% DMSO, 1 μmol/l for 10 min in superfusate). In arterioles, the increase in P_s with TNF-α was not significantly different from P_s with TNF-α + BIM. In venules, the increase in P_s with TNF-α was attenuated significantly by BIM. B: WT vessels (control or TNF-α activated) treated with PP2 (Src blocker in 0.01% DMSO, 2 μmol/l for 10 min in superfusate). P_s in controls was unchanged with PP2, whereas PP2 significantly inhibited the TNF-α-induced increase in P_s. *Significantly different (P < 0.05) from each other; x is significantly different from P_s in TNF-α-treated WT mice (n = 9 vessels; Fig. 1A).

Fig. 7.

Proposed model of ICAM-1-mediated changes in P_s in microvasculature. Under control conditions, there is a constitutive permeability pathway mediated via basally expressed ICAM-1 (independently of interactions with leukocytes; left) in a PKC-dependent manner. Following TNF-α treatment, the expression of ICAM-1 and consequent interactions with β₂-integrins increase, resulting in ICAM-1 clustering and, hence, increasing P_s via a switch to a different, Src-dependent pathway. PMN, neutrophil; TNFR, TNF receptors; PM, plasma membrane.