Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro: a role for transforming growth factor beta (TGF-beta)

Abstract

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([(3)H]thymidine) and collagen synthesis ([(3)H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56 degrees C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor beta (TGF-beta) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-beta neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-beta. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-beta. These data corroborate the importance of eosinophils in skin and lung fibrosis.

Figure 1

Different effects of eosinophils on collagen synthesis in human lung and dermal fibroblasts. Confluent monolayers of lung (A) and dermal (B) fibroblasts were incubated with different concentrations of eosinophil sonicate for 24 hr, and collagen production was evaluated by [³H]proline incorporation. Data are the mean ± SEM of three experiments performed in triplicate or quadruplicate.

Figure 2

Eosinophils contain MMPs but do not affect human lung or dermal fibroblast MMP production. Eosinophil sonicate was added to wells without fibroblasts (I) or to fibroblast cultures (II) at concentrations of 3 × 10⁶ cells per well in DMEM containing 0.1% BSA for 48 hr. The culture supernatants were collected and analyzed by gelatin zymography. (I) Eosinophil sonicate obtained from eosinophils of four different patients was analyzed (lanes A–D). As controls on the same gel, the supernatant of HT1080 cells (lane E) and commercially available pro-MMP-2 were analyzed (lane F). Molecular weight (MW) standards of high-MW range (lane G) and low-MW range (lane H) also were run. (II) Eosinophil sonicate of a fifth patient (lane A), lung fibroblasts (lane B), or dermal fibroblasts (lane D) was incubated alone or eosinophil sonicate was added to either lung fibroblasts (lane C) or dermal fibroblasts (lane E).

Figure 3

Eosinophils contain TIMP-1 and TIMP-2 but do not stimulate TIMP production by dermal fibroblasts. Eosinophil sonicate (lanes: A, 0; B, 10; C, 100; D, 1,000; E, 10,000; or F, 100,000 eosinophils) was added to the lattices containing 100,000 dermal fibroblasts in DMEM containing 10% FCS. On day 7, culture medium was changed in DMEM containing 0.1% BSA and the lattices were incubated further for 48 hr. Culture medium then was collected and analyzed by reverse zymography. Eosinophil sonicate (100,000) was analyzed on the same gel (lane G). The migration positions of control TIMP-1 and TIMP-2 are indicated.

Figure 4

Anti-TGF-β antibodies suppress the stimulation of collagen lattice contraction induced by the eosinophil. Dermal fibroblasts were seeded in collagen lattices (100,000 cells per lattice) and incubated in DMEM + 0.5% FCS alone (solid lozenges) or supplemented with the eosinophil sonicate (10 eosinophils per lattice, ■) or with the eosinophil sonicate preincubated with rabbit anti-TGF-β antibodies (crosses). Open triangles are controls consisting of the eosinophil sonicate preincubated with nonimmune rabbit IgG. Each set of data is the mean of four experiments. SDs were less than 10% of the mean value in every case and were not reported as error bars on the graph for better clarity.