Production of TNF-a, IL-1b, IL-12 and IFN-g in murine peritoneal macrophages on treatment with wheat germ agglutinin in vitro: involvement of tyrosine kinase pathways Ajit Sodhi & Varun Kesherwani Received: 11 January 2007 / Revised: 30 May 2007 / Accepted: 5 June 2007 / Published online: 1 August 2007 # Springer Science + Business Media, LLC 2007 Abstract Treatment of macrophages with various doses of wheat germ agglutinin (WGA) for different time intervals resulted in enhanced expression of TNF-a, IL-1b, IL-12 and IFN-g. The maximum expressions were observed at 24 h with 100 ng/ml of WGA. Enhanced transcription of cytokines TNF-a, IL-1b, IL-12, and IFN-g was observed at 16 h of WGA treatment by RT-PCR. Pharmacological inhibitor of tyrosine kinase, PI3 kinase, protein kinase C, p42/44, p38, JNK and intracellular calcium immobilizing agent down regulated the WGA induced expression of cyto- kines TNF-a, IL-1b, IL-12 and IFN-g. Maximum protein tyrosine kinase activity in macrophages was seen at 5 min of WGA treatment. Maximum cytosolic Ca ++ was observed at 10 min of WGA treatment. WGA treated macrophages showed maximum activation of protein kinase C (PKC) and PI3 kinase at 10 min, p42/44, p38 at 15 min and JNK at 30 min. Transcription factor ELK1 was activated at 60 min and IêB, c-Fos and c-Jun at 30 min of WGA treatment. The pharmacological inhibitors were also used to check the cascade of activation of tyrosine kinase, PKC, PI3 kinase, p42/44, p38, JNK and release of calcium from intracellular storage to sort out the signal pathways involved in the release of TNF-a, IL-1b, IL-12, and IFN-g by macrophages on treatment with WGA in vitro. Keywords WGA . Macrophages . Cytokines . Protein tyrosine kinase Introduction Many macrophage functions involve lectin carbohydrate interaction e.g. adhesion to the endothelium initially relies on the action of selectins (carbohydrate binding proteins) that mediate the attachment of leukocytes to endothelial carbohydrates [1] and recognition and phagocytosis of bac- teria in the absence of opsonins most often involve microbial lectins binding to macrophages glycoconjugate receptors, a process called lectinophagocytosis [2]. Plant lectins also possess biological activities and have been used to study variety of biological processes. Many plant lectins are toxic, some may aggregate erythrocytes of different blood groups (hemagglutination), some are associated with colitis, chronic disease celeaic spruce, IBS (intestinal bowel syndrome) and gut permeability [3] and yet others function as mitogens of T and B lymphocytes [4–6]. In relation to neutrophils lectins with different carbohydrate specificities e.g. ConA binding to mannose and WGA binding to N-acetyl-D-glucosamine (GlcNAc) and sialic acid have been shown to induce cel- lular responses [7–10]. WGA was found to stimulate superoxide anion and H 2 O 2 production by both paraffin oil and thioglycolate elicited mouse peritoneal macrophages [ 11–13] and polymorphonuclear cells [14]. WGA induced significant tumoricidal activity in the tumor associated macrophages at different states of activation [ 15]. Macrophages constitute the major group of phagocytic leukocytes and are crucial to immunosurveillance against invading pathogens and malignancies. Upon activation, macro- phages express one or more cytotoxic effector molecule such as peroxidase, cytolytic protease, nitric oxide (NO) and proinflammatory cytokines [16–19]. The production of proinflammatory cytokines by macrophages is one of Glycoconj J (2007) 24:573–582 DOI 10.1007/s10719-007-9054-z A. Sodhi (*) : V. Kesherwani School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India e-mail: ajit.sodhi@lycos.com