PLoS ONE 10

PLoS ONE 10.1371/journal.pone.0085211 [PMC free article] [PubMed] [CrossRef] [Google Scholar] 43. role in preserving several levels of PTP1B-IN-3 epithelial cell integrity. Keywords: Cell Invasion, Chemokine, Confocal Microscopy, Cytokine Induction, Epithelial Cell, Host-Pathogen Interaction Introduction Mucosal surfaces constantly exposed to a large variety of pathogens are protected by multilayer defense mechanisms. Among these, specific humoral mucosal immunity is dominated by secretory antibodies (Abs)3: secretory immunoglobulin A (SIgA) and secretory immunoglobulin M (SIgM). Both secretory Abs result from the transport across the epithelium of J chain-containing polymeric IgA (mostly dimeric) and pentameric IgM by the polymeric immunoglobulin receptor and exert a role of neutralizing Abs (1, 2). The role of specific SIgA at mucosal surfaces has been studied extensively, and its multiple functional facets extend from immune exclusion to homeostatic control of epithelial integrity (3, 4). As the most conserved Ab among vertebrate species, the importance of IgM has been appreciated for several decades. It is present early in immune development (5) and is known to be crucial in the primary mucosal immune response (6). Moreover, IgM is able to compensate for the lack of IgA in IgA-deficient PTP1B-IN-3 individuals (7). and studies have established the potential of specific, antigen-induced IgM in the systemic neutralization of viruses (8,C10), bacteria (11,C13), fungi (14), and parasites (15,C17). Important advances have come especially from the use of IgM-deficient mice (18), which exhibited a high sensitivity to bacterial and viral infections (19), a condition that could be partly controlled upon administration of normal mouse immune serum (8). Immunotherapy on the basis of the passive administration of human plasma-derived IgG has been used for three decades in clinical applications, with improvement of a large panel of disease conditions like immunodeficiencies, infections, or autoimmune diseases (20, 21). Preclinical and clinical studies have underscored the efficacy against various infectious agents of polyclonal IgM-enriched preparations administered by the systemic route (22,C26). Similar to SIgA, SIgM can be seen as a valid candidate immunoglobulin for mucosal application, given its ability to bind antigens with strong avidity, its potential to ensure long term protection (27), its capacity to survive low pH conditions (28), as well as its resistance to proteases (29). We have demonstrated recently that human plasma can serve as a source of polyreactive, polymeric IgA (pIgA) and IgM to generate secretory-like IgA and IgM Abs, the natural molecular form found in secretions. We found that plasma-derived purified pIgA and IgM can associate with a recombinant secretory component (SC) with a 1:1 stoichiometry and that this association delayed the degradation of pIgA or IgM by intestinal PTP1B-IN-3 washes containing proteases. In addition to these essential biochemical features, we showed that pIgA and secretory-like IgA delayed damage to epithelial polarized Caco-2 cell monolayers induced by a virulent strain of enteropathogenic (29). However, how the plasma-derived Ab operates to block the bacterium and contributes to epithelial homeostasis was not addressed in this study. To provide answers to these open questions, we here dissect the mechanisms of protection conferred by plasma-derived pIgA and secretory-like IgA and then extend this study by evaluating the functionality of human plasma IgM and secretory-like IgM in the same experimental setting. We found that IgM or secretory-like IgM demonstrates a superior ability to maintain transepithelial electrical resistance (TER) and to forestall damage of cell monolayers resulting from infection when compared with pIgA or secretory-like IgA. Bacterial aggregates formed with both plasma pIgA and secretory-like IgA. This phenomenon was amplified upon association with IgM and secretory-like IgM, consistent with the capacity of all polyreactive Ab molecules to recognize virulence factors invasion plasmid antigen (Ipa) B and IpaC, overall suggesting a dual mode of action of the Abs, combining disabling of the bacteria and shielding of the target epithelium. EXPERIMENTAL PROCEDURES Preparation of Human Plasma IgA-, IgM- and IgG-enriched Fractions IgA and IgM were purified from process intermediates of immunoglobulins manufactured from human plasma (30) by affinity chromatography using CaptureSelect human IgA and CaptureSelect human IgM resins (Bioaffinity Co.). The starting material used was a chromatographic side fraction consisting of the strip fraction from an ion exchange chromatography column used in the large scale manufacture of Pdgfd IgG from human plasma. The starting material was diluted in PBS to a target protein (IgA or IgM) concentration of 1 1 mg/ml and then loaded PTP1B-IN-3 onto a CaptureSelect human IgA or IgM column.