Differential cytokine response in interstitial fluid in skin and serum during experimental inflammation in rats

Abstract

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are important mediators produced during inflammation. We hypothesized that the pro-inflammatory cytokine response in the interstitial fluid (IF) is different from that in serum, and we aimed at quantifying the amount of TNF-alpha and IL-1beta in the IF. By centrifugation of rat skin at < 424 g pure IF is extracted. Using ELISA such fluid was analysed for cytokines in back and/or paw skin of pentobarbital-anaesthetized rats, after either induction of endotoxaemia or ischaemia-reperfusion (I/R) injury. During endotoxaemia, TNF-alpha increased in the IF from 0 in control to 640 +/- 100 pg ml(-1) (mean +/-s.e.m.) after 90 min, with the serum concentration being 5-10 times higher at all time points. The response pattern of IL-1beta after lipopolysaccharide (LPS) challenge differed greatly from that of TNF-alpha with a large increase in IF from 390 +/- 90 to 28 000 +/- 1500 pg ml(-1) after 210 min, and a significantly smaller increase in serum (600 +/- 45 pg ml(-1)). During reperfusion of the hind paw after 2 h of ischaemia, there was a gradual increase of TNF-alpha in both IF of the paw skin and serum after 3 min of reperfusion. Both declined after 20 min. The pattern for IL-1beta differed, increasing significantly less in serum (25 +/- 15 pg ml(-1) after 20 min of reperfusion) than in the IF (1100 +/- 200 pg ml(-1)). Immunostaining of the inflamed tissues showed increased expression of the two cytokines in cells of both epidermis and dermis compared to controls. Subdermal injections of TNF-alpha and IL-1beta at the same concentrations found in IF after LPS infusion affected interstitial fluid pressure significantly. Local TNF-alpha production dominates after I/R injury, whereas in endotoxaemia systemic production predominates. For IL-1beta local production dominates in both conditions. Thus, there is a differential pattern of cytokine production and the current method allows the study of the role of cytokines in IF during different inflammatory reactions.

Figure 1. Concentration of TNF-α in serum (•) and interstitial fluid (○) in experimental endotoxaemia, induced by i.v. injection of lipopolysaccharide

The experiment was ended at 30, 90 and 210 min. n = 7 for each time point, except control (0 min, includes serum and IF) where n = 12. * and †, P < 0.001 compared with respective control values. Data are means ± s.e.m.

Figure 2. Concentration of IL-1β in serum (•) and interstitial fluid (○) before and during experimental endotoxaemia (i.v. LPS)

The experiment was ended at 30, 90 and 210 min. n = 7 for each time point, except control (0 min) where n = 12. * and †, P < 0.05 and **P < 0.001 compared with respective control values. Data are means ± s.e.m.

Figure 3. Concentration of TNF-α in serum (○) and interstitial fluid in skin of rat paw (•) during ischaemia and reperfusion injury

n = 7 for each time point. * and †, P < 0.05 compared to respective control values (before start of ischaemia, includes serum and IF). // indicates the 2 h ischaemic period. Data are means ± s.e.m.

Figure 4. Concentration of IL-1β in serum (•) and interstitial fluid in skin of rat paw (○) before ischaemia, after ischaemia and after ischaemia–reperfusion injury

n = 7 for each time point. *P < 0.001 and †P < 0.05 compared to respective control values (before start of ischaemia). // indicates the 2 h ischaemic period. Data are means ±s.e.m.

Figure 5. Microphotographs of immunohistochemical staining of backskin in untreated rats, and in rats following LPS challenge

Increased expression of either IL-1β or TNF-α is represented as red staining. A and B are from untreated/control rats. C and D are stained for IL-1β and photographs E and F are stained for TNF-α 210 min after LPS challenge.

Figure 6. Effects of subdermal injections of TNF-α and IL-1β (1 and 25 ng ml⁻¹, respectively) in paw with (▾, n= 6) or without (•, n= 6) circulatory arrest

Controls (0, n = 8) received 0.9% NaCl. *P < 0.02 compared with own control, with the other paw at the same period or the NaCl group at the same time. Data are means ±s.e.m.

Figure 7. Ratio of interstitial fluid and serum concentrations

A, ratio of concentration of TNF-α and IL-1β in interstitial fluid over serum in early (30 min, black bar) and late (210 min, grey bar) phase of the endotoxaemia. Data are means ±s.e.m.B, ratio of concentration of TNF-α and IL-1β in interstitial fluid over serum in early (3 min, black bar) and late (20 min, grey bar) phase of ischaemia–reperfusion injury. Early phase of IL-1β is missing due to undetectable amounts in serum. Data are means ±s.e.m.