alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation

Abstract

1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

Figure 1

Flow cytometric analysis of the binding of the anti-α₄ integrin mAbs 2B4 (A) and Max 68P (B) to guinea-pig eosinophils. The isotype matched myeloma protein MOPC21 was used for control binding.

Figure 2

Effect of anti-α₄ integrin mAbs on allergen-induced recruitment of ¹¹¹In-eosinophils in guinea-pig skin. 2B4, Max68P or MOPC21 as a control were administered (all at 3 mg kg⁻¹) i.v. 15 min before i.v. injection of ¹¹¹In-eosinophil and i.d. injection of antigen (3, 10 and 30 μg OA, shown as PCA). Sensitized skin sites also received an i.d. injection of saline (shown as saline). Accumulation of ¹¹¹In-eosinophil in sites was assessed after 2 h. Values are mean±s.e.mean of experiments in six animals with each mAb. ^*P<0.05 compared to responses in MOPC21 treated animals.

Figure 3

Effect of anti-α₄ integrin mAbs on recruitment of ¹¹¹In-eosinophil induced by ZAP or arachidonic acid (AA) in guinea-pig skin. 2B4 (a) or Max68P (b), and MOPC21 as a control, were administered (all at 3 mg kg⁻¹) i.v. 15 min before i.v. injection of ¹¹¹In-eosinophil and i.d. injection ZAP (3, 10 and 30% dilution in saline) or AA (30 nmol). Accumulation of ¹¹¹In-eosinophils in sites was assessed after 2 h. Values are mean±s.e.mean of experiments in six animals with each mAb.

Figure 4

Effect of combined treatment with anti-α₄ and β₂ integrin mAbs on (a) allergen-induced and (b) ZAP-induced ¹¹¹In-eosinophil accumulation in guinea-pig skin. Approximately 15 min before the i.v. injection of ¹¹¹In-eosinophils, animals were treated with an i.v. injection of either control mAb (MOPC21, 3.25 mg kg⁻¹), anti-α₄ integrin (2B4, 3 mg kg⁻¹), anti-β₂ integrin mAb (6.5E, 0.25 mg kg⁻¹) or α₄ and β₂ mAbs in combination. Allergen (OA, 3 – 30 μg), ZAP (3 – 30%) or saline were injected i.d. and the ¹¹¹In-eosinophil accumulation per skin site was assessed 2 h later. Results are expressed as mean±s.e.mean of five animals for each treatment group. ^*P<0.05 compared to responses in MOPC21 treated animals.

Figure 5

Effect of pretreating skin sites with antiserum or LPS on the capacity of anti-α₄ integrin mAb 2B4 to module ZAP- and PAF-induced ¹¹¹In-eosinophil accumulation in guinea-pig skin. Skin sites were pretreated for 16 – 20 h with antiserum (AS, 50 μl of 1 in 30 dilution) or LPS (0.0125 ng) followed by i.d. injection of OA (to induce the PCA reaction), ZAP (10% in saline) or PAF (1 nmol). 2B4 or MOPC21 were administered (3 mg kg⁻¹) i.v. 15 min before i.v. injection of ¹¹¹In-eosinophil. Accumulation of ¹¹¹In-eosinophils in sites was assessed after 2 h. Values are mean±s.e.mean of experiments in four animals per group. ^*P<0.05 compared to responses in MOPC21 treated animals.

Figure 6

Effect of pretreatment of ¹¹¹In-eosinophil with anti-α₄ integrin mAb 2B4 on their capacity to migrate to inflammatory sites in guinea-pig skin. Radiolabelled eosinophil were incubated with MOPC21 or 2B4 (both at 50 μg ml⁻¹), washed and infused into separate recipient guinea-pigs. Sensitized skin sites were injected with antigen (30 μg OA, shown as PCA), ZAP (10% in saline) or PAF (1 nmol) and accumulation of ¹¹¹In-eosinophil in sites assessed after 2 h. Values are mean±s.e.mean of six experiments.