Direct measurement of cytokines (IFN-gamma, IL-4, -5, and -6) from organs after antigenic challenge.

A mouse in vivo model has been developed in which cytokines produced within the regional lymph nodes and other organs after an antigenic challenge were measured directly without the need of an in vitro reculturing step. Mice were subcutaneously (s.c.) immunized with antigens such as keyhole limpet hemocyanin (KLH) in aluminium hydroxide (alum) at the base of the tail. After defined periods of time lymph nodes, spleen, liver, and lung were collected and frozen. The organs were then homogenized (or sonicated), centrifuged, and the cytokine levels, for example, IFN-gamma, IL-4, IL-5, and IL-6 were determined by ELISA. It was found that injection of alum alone caused measurable cytokine production in draining lymph nodes, but inoculation of an antigen-alum mixture, such as KLH and dust-mite antigen (DMA), significantly increased in vivo cytokine production over that of alum alone. In the dose range tested there appeared to be an inverse relationship between the dose of antigen and cytokine levels. Thus, 0.1-microgram KLH induced more IL-4 in vivo than 10-micrograms KLH. Kinetic studies showed that for IL-4 and IFN-gamma, peak production occurred around days 5 and 6, respectively. Route of immunization and the dose of antigen were found to be very critical, in that injection of 10-micrograms DMA intraperitoneally induced significant levels of cytokines in the lung, while the same dose of antigen given at the base of tail, s.c., did not induce appreciable levels of cytokines in any organ tested. Cyclosporin A inhibited in vivo production of IFN-gamma, IL-4, and IL-5 by approximately 80%, but not that of IL-6. Surprisingly, dexamethasone enhanced the production of IL-6 while inhibiting all the other cytokines. In conclusion, these results suggest that direct determination of cytokines in the organs may provide an easy readout to assess differential effects of immunoregulatory molecules on the production of Th1- and Th2-type cytokines in vivo.