IL-4 Expression and Constitutive Activation of Erk Signaling In Vivo in Follicular Lymphoma.

Follicular lymphoma (FL) is characterized by constitutive expression of the Bcl-2 oncoprotein as a consequence of the t(14;18) BCL2/JH translocation in germinal center B-cells. While Bcl-2 confers survival advantage through anti-apoptosis mechanisms, the t(14;18) alone is not sufficient for lymphomagenesis. Evidence suggests that additional factors in the lymph node microenvironment, related to tumor infiltrating T-cells and macrophages, may cooperate with Bcl-2 in orchestrating the malignant phenotype in vivo. In this study we generated proteomic cytokine profiles of patient lymph node tissue lysates from FL (N=53) grades 1–3, and the non-malignant normal counterpart follicular hyperplasia (FH; N=24). Using an ultrasensitive multiplex elisa system we assayed a panel of 11 cytokines known to be secreted by T-cells and/or macrophages including IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12p70, TNF-alpha, and INF gamma. With the exception of IL-4, all cytokines measured showed higher concentrations in FH than FL. In particular, IL-1 beta demonstrated levels 20 times higher in FH; IL-8 levels were 30 times higher; and IL-13 levels were 9 times higher in FH than in FL (p values < 0.005). In contrast, IL-4 levels were 5 times higher in FL than in FH (p < 0.0054). Interestingly, IL-4 levels were highest in FL grade 1 (N=20) in comparison to grade 2 (N=18) and grade 3 (N=12). Immunohistochemical stains for CD3 showed no significant difference in the numbers of CD3+ T-cells in FL samples over FH lymph nodes; or in FL grade 1 vs. grade 2 or grade 3. Hence the differences observed are not due to numerical differences in the total number of T-cells present in FL and FH. We evaluated the activation state of the mitogen activated protein (MAP) kinase signaling pathway, which is activated in response to stimulation by most cytokines in our panel via PI3Kinase. Western blot analysis showed that Erk was constitutively phosphorylated in 14 out of 15 (93%) of FL cases in contrast to only 3 out of 9 (33%) of FH cases. These findings demonstrate that tumor growth in FL is characterized overall by low levels of cytokines within the lymph node microenvironment in comparison to growth associated with the benign counterpart FH. The T-cell secreted cytokine, IL-4, which drives B-cell proliferation, was the only cytokine that showed statistically significant higher expression in the lymph node microenvironment of FL in comparison to FH. Moreover, the intracellular MAP kinase signaling pathway was constitutively activated in vivo in patient FL samples as compared to FH, despite higher overall cytokine levels in FH. These findings suggest that the IL-4 cytokine, and intracellular Erk activation in FL cells, may 1) play a role, in cooperation with Bcl-2, in driving the malignant phenotype in FL and 2) may serve as effective molecular targets for future therapies in FL.