Eosinophil recruitment in type-2 hypersensitivity pulmonary granulomas: source and contribution of monocyte chemotactic protein-3 (CCL7)

Abstract

Monocyte chemotactic protein-3 (MCP-3/CCL7) has potent eosinophil chemoattractant properties. The present study determined its relative contribution to the formation of Th2 cytokine-mediated (type-2) eosinophil-rich interstitial lung granulomas induced by antigens of Schistosoma mansoni eggs. Both MCP-3 transcripts and protein levels were more strongly expressed in lungs with type-2 than with type-1 (mycobacterial antigen-elicited Th1-mediated) granulomas. In vivo treatment with neutralizing antibodies demonstrated that MCP-3 abrogated eosinophil accumulation in type-2 lesions by 40 to 50%. Immunohistochemical staining revealed that MCP-3 localized to vessels in or near granulomas suggesting that endothelial cells were an important in situ source of MCP-3. Maximal MCP-3 transcript expression was abrogated by anti-interleukin-4 treatment. Furthermore, cultured mouse lung endothelial cells displayed augmented MCP-3 production in response to interleukin-4. Together, these results suggest that MCP-3 contributes to a significant component of eosinophil recruitment in the type-2 interstitial granuloma formation and Th2 cytokines promote its production.

Figure 1.

Enhanced MCP-3 (CCL7) expression during pulmonary, type-2 (SEA) bead granuloma formation. A: Relative component of eosinophils in dispersed type-1 (PPD) and type-2 (SEA) bead granulomas. Bars are means ± SD of three to four separate experiments. B: MCP-3 protein and mRNA expression in granulomatous lungs. 0 = prechallenge lung. Bars are means ± SD from two experiments with five mice per point in each experiment.

Figure 2.

Histological appearance of type-2 (SEA) bead granulomas in mice treated with antibodies to eotaxin and MCP-3. Type-2 (SEA) lesions were generated in presensitized CBA mice, then at the time of bead challenge they were administered 10 mg of purified IgG (nonimmune rabbit, anti-eotaxin, anti-MCP-3, or 10 mg each of anti-eotaxin plus anti-MCP-3 Abs). On day 4 after Ab treatment and bead challenge, lung tissues were harvested and histological sections were prepared as described in Materials and Methods. A, Nonimmune IgG treated; B, anti-MCP-3 treated; C, anti-eotaxin treated; D, combined anti-MCP-3 and anti-eotaxin treatment. Granulomas from each of the five to six mice per treatment group were analyzed morphometrically and the inset numbers indicate mean eosinophil counts per 50 × 50 μm area ± SD. Four such areas were measured from each granuloma. Original magnifications: ×200 (A to D); ×400 (insets).

Figure 3.

In vivo IL-4 neutralization abrogates MCP-3 (CCL7) transcript expression and eosinophil recruitment in lungs with type-2 (SEA) bead granuloma formation. Just before bead challenge mice were treated with anti-cytokine Abs as described in the Materials and Methods, then 4 days later lungs were harvested for analysis. A: MCP-3 (CCL7) transcript expression. Bars are means ± SD of three experiments. B: Relative component of eosinophils in isolated, dispersed granulomas. Bars are means ± SE derived from two experiments with a total of six to eight mice per group.

Figure 4.

Immunohistochemical localization of MCP-3. Frozen sections of lungs were prepared and stained with anti-MCP-3 Abs as described in Materials and Methods. Note staining of vessels (V) in A, C, and E. A: Type-2 (SEA) granulomas, MCP-3 stain. B: Serial section of A, nonimmune Ab control. C: Vessel in proximity to type-2 (SEA) granuloma, MCP-3 stain. D: Serial section of C, nonimmune Ab control. E: Type-2 (SEA) bead granuloma, MCP-3 stain, low-power magnification. F: Serial section of E, nonimmune Ab control. G: Parenchyma and vessel in normal lung, MCP-3 stain. H: Control bead lesion in lung challenged with antigen-free beads, low magnification. Original magnifications: ×200 (A, G);×400 (C); ×100 (D, E, H).

Figure 5.

Immunohistochemical localization of eotaxin. Frozen sections of lungs were prepared and stained with anti-eotaxin Abs as described in Materials and Methods. A: Bronchus from lung with type-2 (SEA) granulomas, eotaxin stain. B: Serial section of A, nonimmune Ab control. C: Lung parenchymal alveoli in lung with type-2 (SEA) granulomas, eotaxin stain. D: Lung parenchymal alveoli in lung with type-2 (SEA) granulomas, nonimmune Ab control; E, Type-2 (SEA) bead granuloma, eotaxin stain. F: Bronchus and nearby control bead lesion in lung challenged with antigen-free beads. Original magnifications: ×200 (A, C, F); ×400 (D, E).

Figure 6.

Interleukin-4 induced MCP-3 (CCL7) production by lung endothelial cells. Lung endothelial cells were cultured with graded doses of IL-4 as described in the Materials and Methods and then 24 hours supernates were assayed for MCP-3 and eotaxin by ELISA. Bars are means ± SD of triplicate cultures.