Tumor necrosis factor-alpha from macrophages enhances LPS-induced clara cell expression of keratinocyte-derived chemokine

Abstract

Tumor necrosis factor (TNF)-alpha is a cytokine produced by alveolar macrophages in response to LPS in the lung. Clara cells are bronchiolar epithelial cells that produce a variety of proinflammatory cytokines in response to LPS but not to TNF-alpha. In this study, we examined whether TNF-alpha affects Clara cell cytokine production in the setting of LPS stimulation. Using a transformed murine Clara cell line (C22), we observed that both LPS and TNF-alpha induced production of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein (MCP)-1. We also found that simultaneous LPS and TNF-alpha stimulation is synergistic for KC production, but additive for MCP-1 production. By using a Transwell coculture system of RAW264.7 macrophages and Clara cells isolated from C57Bl/6 mice, we found that macrophages produce a soluble factor that enhances Clara cell KC production in response to LPS. Cocultures of Clara cells from mice deficient in TNF-alpha receptors with RAW264.7 macrophages demonstrated that the effect of macrophages on Clara cells is mediated primarily via TNF-alpha. To determine whether these findings occur in vivo, we treated wild-type and TNF receptor-deficient mice intratracheally with LPS and examined the expression of KC. LPS-treated, TNF receptor-deficient mice showed much less KC mRNA in airway epithelial cells compared with wild-type mice. In contrast, a similar number of KC-expressing cells was seen in the lung periphery. Thus, upregulation of KC by Clara cells in the setting of LPS stimulation is largely dependent on TNF-alpha originating from alveolar macrophages. These findings shed light on macrophage-Clara cell interactions in regulating the pulmonary inflammatory response to LPS.

Figure 1.

Cytokine profile of C22 cells in response to tumor necrosis factor (TNF)-α. C22 cells were treated with 20 ng/ml TNF-α for 24 hours. The conditioned media from three independent experiments were collected, and cytokine production was assayed using an antibody array. Blots were scanned, optical densities adjusted based on positive controls (upper left, lower right), and the fold change, from untreated to TNF-α–treated, calculated as described in Materials and Methods. A representative blot is shown with its quantification. Solid ellipses, keratinocyte-derived chemokine (KC); long black dashed ellipses, monocyte chemoattractant protein (MCP)-1; long gray dashed ellipses, regulated upon activation, normal T-cell expressed and secreted (RANTES); short black dashed ellipses, TNF-α.

Figure 2.

KC and MCP-1 secretion by C22 cells in response to LPS or TNF-α. C22 cells were treated with various concentrations of LPS (A) or TNF-α (B) for 24 hours, and the levels of KC (open bars) and MCP-1 (shaded bars) in the conditioned media were measured by ELISA. Alternatively, C22 cells were treated with 10 ng/ml LPS (C) or with 25 ng/ml TNF-α (D) for various times up to 24 hours. Cells treated with serum-free media alone served as control. Solid triangles, KC control; solid squares, KC treated; shaded triangles, MCP-1 control; shaded squares, MCP-1 treated. Data represent means (± SEM) of at least three independent experiments done in triplicate.

Figure 3.

Secretion of KC by C22 cells in response to combined LPS and TNF-α is synergistic. C22 cells were treated with 10 ng/ml LPS alone, 25 ng/ml of TNF-α alone, or LPS and TNF-α combined for 24 hours and the levels of KC (open bars) and MCP-1 (shaded bars) in the conditioned media were measured by ELISA. Data represent means (± SEM) of at least three independent experiments done in triplicate. *P < 0.05.

Figure 4.

The effect of macrophages on the Clara cell secretion of KC to LPS is mediated via TNF-α. Freshly isolated Clara cells from wild type (WT) (A) or TNF receptor knockout (TNFR-KO) (B) mice were placed in the bottom chamber of a Transwell system alone or with RAW264.7 cells in the upper chamber, as described in Materials and Methods, and were treated with 10 ng/ml LPS for 24 hours. The levels of KC in the conditioned media were measured by ELISA. Data represent means (± SEM) of at least three independent experiments done in triplicate. *P < 0.05.

Figure 5.

The number of KC-expressing cells is significantly reduced in distal airways of TNFR-KO mice compared with wild-type (WT) mice in response to LPS. WT (A–C) and TNFR-KO (D–F) mice were given PBS alone (A and D) or containing 5 μg/ml LPS (B–C, E–F) by intratracheal injection. After 2 hours, the lungs were inflation fixed and paraffin embedded, and lung sections were examined for KC expression by in situ hybridization. Images represent at least three independent experiments using at least two mice per condition. The number of KC-expressing cells was determined by counting the number of digoxigenin-positive cells along 200 μm of basement membrane (BM) of distal airways from five lung sections per mouse (G) or within the peripheral lung (H) in five high-power fields (HPF) of one lung section from at least seven mice per condition. Data represent means (± SEM). *P < 0.05.

Figure 6.

Macrophage-derived TNF-α augments Clara cell responses to LPS via TNFR1. RNA isolated from C22 cells transfected with TNFR1, TNFR2, or scrambled (Scr) small interfering RNAs were treated with LPS alone (open bars) or in coculture with RAW264.7 macrophages (shaded bars). The RNA was reverse transcribed and quantitative PCR reactions were performed for KC. Data represent means (± SEM) of at least four independent experiments done in duplicate. *P < 0.05; NS = not significant.