“PERFEXT”: a direct method for quantitative assessment of cytokine production in vivo at the local level

Abstract

A method termed "PERFEXT", based on sequential perfusion and detergent extraction of lymphoid and non-lymphoid organs, has been developed for the quantitative measurement of cytokines produced at a local level in a given tissue. In vivo treatment of mice with Staphylococcus enterotoxin B (SEB) or lipopolysaccharide (LPS) served as the model systems. Interleukin-2 (IL2) and interferon-gamma (IFN gamma) levels were monitored by ELISA analysis of extracted samples. After local footpad (FP) injection with SEB, spleen and serum IL2 levels peaked at 2-4 h, while IL2 levels peaked at around 4-8 h in both FP and popliteal lymph nodes. SEB injection resulted in increased IFN gamma levels both in the FP and the draining lymph node. The detection of cytokines in the intestine allows for the application of the method at mucosal sites as well, provided enzyme inhibitors are present during the extraction procedure. After FP injection with LPS, IFN gamma production was significantly increased in the draining lymph node and was detectable in the FP, whereas IL2 was undetectable in any organ examined. IL2 and IFN gamma could also be detected at the site of elicitation of a delayed-type hypersensitivity reaction following local FP challenge. Local cytokine production correlated with the swelling response, whereas cytokine production in the spleen did not. IL2 peaked early, followed by a late increase in IFN gamma production, corresponding to the maximum swelling. This simple method should prove useful for analysing the production of other cytokines in vivo in distinct anatomical compartments.