Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes Isabelle Bekeredjian-Ding1*, Sandra Foermer1, Carsten J. Kirschning 2 , Marijo Parcina1, Klaus Heeg1 1 Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, Heidelberg, Germany, 2 Institute for Medical Microbiology, University Hospital Essen, Essen, Germany Abstract Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations. Citation: Bekeredjian-Ding I, Foermer S, Kirschning CJ, Parcina M, Heeg K (2012) Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes. PLoS ONE 7(1): e29806. doi:10.1371/journal.pone.0029806 Editor: Troy D. Randall, University of Rochester School of Medicine, United States of America Received January 17, 2011; Accepted December 6, 2011; Published January 4, 2012 Copyright: 2012 Bekeredjian-Ding et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: IB-D received financial support from the Olympia-Morata program of the Medical Faculty of the University of Heidelberg, Germany. The Olympia- Morata grant is intramural funding from the medical faculty of the University of Heidelberg. There were no external grants involved. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: isabelle.bekeredjian-ding@med.uni-heidelberg.de Introduction Due to their immunostimulatory properties plant extracts are commonly used for immune therapy, usually with the aim of inducing an immune response in patients considered to be immunodeficient due to an underlying malignancy. However, despite frequent reports of successful outcomes little is known on the exact mechanisms mediating these beneficial effects. In the recent past, many groups have described plant-derived substances with immunostimulatory properties. Among them several reports claimed that their effects were based on the activation of Toll-like receptor (TLR)-4, a pattern recognition receptor that mediates proinflammatory immune responses to Gram negative bacteria due to recognition of the lipid A component of the cell wall-associated lipopolysaccharide (LPS). The nature of these substances ranges from proteins such as a 55 kDa protein identified in Aeginetia indica [1] to polysaccharides as in Acanthopanax senticosus, Acanthopanax koreanum and Carthamus tinctorius L. [2,3,4]. However, many researchers have raised concerns since LPS contamination is easily introduced during the purification procedure, and TLR4 activity may, therefore, not represent an intrinsic property of the plant-derived molecules. Another plant-derived polysaccharide is poke weed mitogen (PWM), a lectin derived from Phytolacca americana. Due to their potent immunostimulatory properties Phytolacca americana extracts are used in patients with infections and cancer. Furthermore, PWM is commonly used for B cell assays in vitro. Along with ‘‘SAC’’, a suspension of formalin-fixed S. aureus cells, it is the most frequently used stimulatory reagent in assays performed for diagnostic purposes, e.g. evaluating B cell function in patients with suspected immunodeficiency. Based on previous studies it is clear that PWM potently stimulates B cell proliferation and immunoglobulin production [5,6]. Furthermore, combination of SAC and PWM results in a synergistic increase in B cell activation and has therefore been proposed as a better control stimulus for B cell assays [5]. Previously, we showed that SAC stimulation results from synergistic effects of S. aureus surface protein A and bacterial Toll-like receptor (TLR) ligands, such as TLR2-active lipoproteins [7]. By contrast, the effects of PWM have been attributed to the crosslinking of glycoproteins on the B cell surface [8,9]. More recently, however, one report claimed that commercially available PWM preparations contain contaminant LPS, which contributes to immune cell activation [10]. However, we previously demon- strated that human peripheral blood B cells lack relevant TLR4 expression and are unresponsive to LPS stimulation [11,12]. Therefore, TLR4 activity cannot readily explain the mitogenic activity of the poke weed lectin on human B cells. In views of its frequent utilization, in the present study we sought to identify its mode of action. Materials and Methods Mice and murine B cell isolation The use of mice was approved by the interfacultary bio- medical research facility and the governmental agency at the PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29806