To the best of our knowledge, this is the first time quantification of cytokines has been done in interstitial fluid in normal and in inflamed skin. Most reports regarding elevated amounts of cytokines in inflammation are described by qualitative methods, or indirectly. The centrifugation method used in this study, and also the wick method, makes it possible to get access to uncontaminated interstitial fluid from skin and muscle. Combined with ELISA, these methods enable us to quantify the amount of different inflammatory proteins in this fluid, and thereby get a better understanding of the mechanisms involved in inflammatory processes occurring locally at the tissue level.
Various techniques have been developed for sampling tissue fluid; all of these have their different limitations (Aukland & Reed, 1993). A potential risk involved in these methods is cell damage, resulting in contamination of the extracellular fluid with intracellular fluid and proteins. In a recent paper we have developed and validated a centrifugation method to isolate interstitial fluid (Wiig et al. 2003). By centrifugation of skin at a G-force < 424 g, we found the contribution from the intracellular compartment to be negligible as examined by 51Cr-EDTA. This probe will not enter the cells (Løkken, 1970). After centrifugation the ratio of 51Cr-EDTA in centrifugate and plasma was not different from 1.0, demonstrating that intracellular fluid was not diluting the fluid obtained by centrifugation. Centrifugation will sample fluid from the whole extracellular fluid volume phase, but the centrifugate was found to contain less than 2% of a tracer confined in the intravascular fluid phase, suggesting that the contribution from this compartment was negligible. Contamination of the skin by blood during the experiment might be a problem, but if this happened (only twice during the whole experiment) the skin was washed with saline and thereafter gently dried. Given the reservations above we feel confident that the present fluid isolation method will reflect the changes taking place in the interstitial fluid during experimental inflammation.
Our results show that in endotoxaemia there is an increase of TNF-α and IL-1β in the interstitium, and that the cytokine level continues to increase throughout a 3 h experimental period for IL-1β, and for at least 90 min for TNF-α. The concentrations of IL-1β in IF were much larger than TNF-α, and even a detectable amount of IL-1β was found in IF in controls. When comparing TNF-α and IL-1β, the TNF-α is substantially higher in serum, while IL-1β is largely confined to the IF. In Fig. 7A this is presented as the ratio of mean concentration in interstitial fluid over serum in an early (30 min) and late (210 min) phase of endotoxaemia. The observed ratio of concentration in IF over that in serum being larger than 1 clearly demonstrates that IL-1β is produced in the interstitium, since any protein being transported across the microvasculature from plasma to IF will be present in lower concentrations in IF than in plasma (Michel & Curry, 1999). In I/R we found increased amounts of TNF-α and IL-1β in skin of paw after the 2 h ischaemic period. This is in agreement with results from ischaemic myocardium (Shames et al. 2002) where ischaemia alone increased TNF-α gene expression and peptide synthesis. Initially there was an increase of TNF-α in both serum and interstitium, where the concentration in interstitial fluid was up to 10 times higher than in the circulation. As evident from Fig. 3, the levels of TNF-α increased to a maximum at around 3 min of reperfusion, followed by a gradual reduction in concentration at continued reperfusion. After only 20 min of reperfusion, TNF-α in interstitial fluid decreased rapidly to levels measured in animals having only ischaemia of the limb, while serum concentrations declined more slowly. This observed reduction in TNF-α in the IF could be due to a washout effect of the cytokine from the interstitium, meaning that the TNF-α in the IF in the reperfused tissue is brought into the circulation via the lymphatics, again explaining why the levels of TNF-α in the circulation have a slower decline than in the IF. Compared to endotoxaemia, the results for I/R states show that the latter is more a local inflammation.
The levels of IL-1β during I/R-injury had a development different from TNF-α. In serum IL-1β did not appear until 20 min of reperfusion, whereas in IF there was an increase already after 3 min of reperfusion, an increase that continued after 20 min of reperfusion. A hypothesis that may be derived from these observations is that IL-1β seen after I/R is largely derived from the interstitium. In Fig. 7B the ratios for IF/serum in an early (3 min) and late (20 min) phase of I/R-injury are presented. The ratio for IL-1β is clearly over 1, again indicating a local production (the early phase is lacking due to undetectable amounts in serum). Similarly, the TNF-α ratios are over 1, indicating that this cytokine also is produced locally during I/R-injury.
Immunhistochemistry revealed that the source of IL-1β in the tissue, in both endotoxaemia and I/R injury, was cells in the epidermis and cells surrounding the hair follicles. The source for TNF-α in the interstitium during endotoxaemia was cells in the basal layer of epidermis, but also fibroblast-like cells in the interstitium. A somewhat different pattern was found in I/R injury, where the increased TNF-α expression was seen in epithelial cells surrounding the hair follicles.
We have previously shown a role for TNF-α and IL-1β in exchange of fluid from the capillaries and into the interstitium by lowering of the interstitial fluid pressure (Pif), and we have also seen a significant lowering of Pif and increased albumin extravasation after I/R injury in hindlimb of rats (Nedrebøet al. 2003), and during endotoxaemia in rats (Nedrebø & Reed, 2002). In the present study we observed that both these cytokines were up-regulated in the IF in both of these inflammatory reactions. Furthermore, by applying the observed concentrations directly to subcutis we were able to elicit a change in Pif mimicking that observed in previous LPS and I/R studies, a response inducing fluid flux across the capillaries. These observations strongly suggest that these cytokines take part in control of fluid exchange during inflammation. TNF-α has been reported to induce increased vascular endothelial permeability (Royall et al. 1989) and tissue oedema formation (Beutler et al. 1985). It has also been reported that alterations of the extracellular matrix, which can reduce cell-matrix contact, could be the result of TNF-α causing increased endothelial permeability (Partridge et al. 1992).
One of the most surprising findings in this study was the elevated amounts of IL-1β in control, i.e. untreated animals. That this observation was not due to a methodological artefact was shown by the lack of IL-1β in unstimulated muscle. IL-1 has been found to be present in normal human epidermis, most likely IL-1α (Hauser et al. 1986). IL-1 is not produced by unstimulated cells, with the exception of keratinocytes in skin, some epithelial cells and cells of the central nervous system (Dinarello, 1994a,b). Normal production of IL-1 is, however, critical for initiation of normal host response to injury and infection. Intracellular IL-1β consists exclusively of a 31 kDa precursor form (Hazuda et al. 1990). Extracellular IL-1β consists of a mixture of both the precursor and the mature IL-1β. There are reports that ELISA kits using mature IL-1β as a standard will detect, but considerably underestimate, the unprocessed IL-1β precursor (Dinarello, 1992; Herzyk et al. 1992). The precursor form is usually not the predominant form of IL-1β. If we compare the response of TNF-α and IL-1β, we observe that the production and large amount of IL-1β are much more confined to the interstitium than are those of TNF-α. The rapid increase of IL-1β could be explained by already existing precursor IL-1β being rapidly activated to the mature form by interleukin-1β-converting enzyme (ICE).
In conclusion, the present experiments show that quantification of inflammatory mediators in plasma/serum does not give a representative picture of concomitant changes in the interstitium. We have demonstrated a fundamental difference in the expression of the two cytokines studied, TNF-α and IL-1β. IL-1β was markedly up-regulated in the interstitium both in a local and systemic inflammatory reaction, while the TNF-α response pattern differed in these two conditions. The cytokine concentrations observed in IF during LPS stimulation were able to induce lowering of Pif, suggesting a mechanistic role of these substances in inflammatory reactions. Isolation of native IF allows us to analyse the fluid in which the communication between cells and extracellular matrix takes place and thereby get a better understanding of the molecular mechanisms involved in inflammatory processes.