VCAM-1 contributes to rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4: evidence for basal expression of functional VCAM-1 in rat skin

Abstract

The aim of the present study was to investigate the role of the adhesion pathway alpha4 integrins/vascular cell adhesion molecule type 1 (VCAM-1) in rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4. For this purpose we have used an in vivo model of local 111In-eosinophil accumulation to quantify eosinophil accumulation induced by intradermal administration of platelet-activating factor (PAF) and leukotriene B4 (LTB4) in rats. Initial experiments carried out over 4 hr demonstrated that intravenous administration of an anti-VCAM-1 monoclonal antibody (mAb; 5F10) or an anti-alpha4 integrin mAb (TA2) caused a significant reduction in PAF- or LTB4-induced 111In-labelled eosinophil accumulation. Time-course experiments demonstrated that the anti-VCAM-1 mAb was effective at suppressing early phases of the 111In-labelled eosinophil accumulation induced by PAF and LTB4 (e.g. within the first 60 min). In contrast, 111In-labelled eosinophil accumulation induced by these chemoattractants was unaffected by the local administration of the transcriptional inhibitor actinomycin D, suggesting a role for basally expressed VCAM-1. Indeed, basal expression of VCAM-1 in rat skin sites was demonstrated by the localization of intravenously administered radiolabelled mAb. The localization of the radiolabelled antibody was not altered in skin sites injected with PAF or LTB4. Finally, the inhibitory effects seen with the anti-VCAM-1 mAb were enhanced when the antibody was co-injected into rats with an anti-intercellular adhesion molecule-1 (ICAM-1) mAb (1A29). The combination of these two mAb also caused a significant inhibition of PAF-induced oedema, as quantified by the local accumulation of 125I-labelled human serum albumin. The results indicate a role for alpha4 integrins/VCAM-1 and ICAM-1, in PAF- and LTB4-induced eosinophil accumulation in vivo and suggest that basally expressed VCAM-1 may have a functional role in rapid accumulation of eosinophils induced by chemoattractants.

Figure 1

Effect of the anti-VCAM-1 mAb 5F10 on PAF- and LTB₄-induced ¹¹¹In-eosinophil accumulation in rat skin. Animals were pretreated with 5F10 (2 mg/kg i.v.) or MOPC-21 (2 mg/kg i.v.) 15 min prior to the i.v. administration of ¹¹¹In-eosinophils. (a) PAF or (b) LTB₄ were administered intradermally and the responses were quantified 4 hr later as described in the Materials and Methods. The results, which have been corrected for the small level of counts in Tyrode-injected skin sites, are expressed as mean±SEM for at least seven pairs of rats. Significant differences in responses detected between MOPC-21-treated and 5F10-treated rats are shown by asterisks, *P < 0·05 and **P < 0·01.

Figure 2

Effect of the anti-rat α₄ integrin mAb TA2 on PAF- and LTB₄-induced ¹¹¹In-eosinophil accumulation in rat skin. Animals were pretreated with TA2 (5 mg/kg i.v.) or MOPC-21 (control, 5 mg/kg i.v.) 15 min prior to the i.v. administration of ¹¹¹In-eosinophils. (a) PAF or (b) LTB₄ were administered intradermally and the responses were quantified 4 hr later as detailed in the Materials and Methods. The results, which have been corrected for the small level of counts in Tyrode-injected sites, are expressed as mean±SEM (n = at least seven pairs of rats). Significant differences in responses detected between MOPC-21-treated and TA2-treated rats are shown by asterisks; *P < 0·05 and **P < 0·01.

Figure 3

Effect of the anti-VCAM-1 mAb 5F10 on time–course of PAF-and LTB₄-induced responses. PAF or LTB₄ (both at 10⁻¹⁰ mol/site) were injected intradermally at different time-points, −3 hr, −1 hr, −0·5 hr and 0 hr, prior to the i.v. administration of the antibodies (5F10 and MOPC-21, both at 2 mg/kg), the ¹¹¹In-eosinophils and ¹²⁵I-HSA. After a 1-hr accumulation period, the animals were killed and the ¹¹¹In-eosinophil accumulation/skin site and oedema formation were quantified as described in the Materials and Methods. Panels (a) and (b) show the time–course profiles of ¹¹¹In-eosnophil accumulation induced by PAF and LTB₄, respectively, and panel (c) shows the time–course of PAF-induced oedema. The results, which have been corrected for the small level of counts detected in corresponding Tyrode-injected skin sites, are mean±SEM for (n = at least five pairs of rats). Significant differences in responses detected between MOPC-21-treated and 5F10-treated rats are shown by asterisks, *P < 0·05 and **P < 0·01.

Figure 4

Effect of locally administered actinomycin D on (a) PAF- or (b) LTB₄-induced ¹¹¹In-eosinophil accumulation in rat skin. PAF and LTB₄ (both at 10⁻¹⁰ mol/site) were injected intradermally with or without actinomycin D (5×10⁻⁹ m/site) at −3 hr, −1 hr, −0·5 hr and 0 hr prior to the i.v. administration of ¹¹¹In-eosinophils. After a 1-hr accumulation period, ¹¹¹In-eosinophil accumulation/site was quantified as detailed in the Materials and Methods. The results, which have been corrected for the small level of counts detected in corresponding Tyrode/actinomycin D-injected skin sites, are mean±SEM for n = at least five pairs of rats.

Figure 5

Uptake of radiolabelled anti-VCAM-1 mAb into rat skin sites. Skin sites were un-injected, or injected intradermally with Tyrode solution, PAF, or LTB₄(both at 10⁻¹⁰ mol/site). At 1 or 4 hr post injections, the animals received an intravenous dose of radiolabelled antibodies including ¹²⁵I-labelled 5F10 (anti-VCAM-1) and ¹¹¹In-labelled control mAb. Five minutes later, the circulation was perfused and the skin sites removed and localisation of antibodies was quantified as described in the Materials and Methods. The graph shows the percentage uptake of the injected dose (ID) of radiolabelled 5F10 per gram of tissue, corrected for the non-specific uptake of radiolabelled control mAb (filled bars, n = 4 rats). Some animals received both the radiolabelled mAb and a 50-fold excess of unlabelled 5F10 (open bars, n = 3 rats). The results are mean±SEM for n number of rats. Asterisk indicates a significant difference from the uptake in uninjected sites, P < 0·05.