3). secretion of polyreactive antibodies. Contamination with a computer virus or tissue damage induced by a toxin similarly led to an increase in polyreactive antibodies in MyD88+/+, but not MyD88/mice. We conclude that activation of TLRs is usually a key link in the mechanism of polyreactive antibody secretion into the blood circulation. Natural antibodies have been known for over 100 years and are now considered part of the innate immune system. Many of these antibodies are polyreactive. That is they are capable of binding to a wide variety of structurally unrelated self (e.g., proteins, lipids, carbohydrates, DNA) and non-self (e.g., bacteria, viruses) antigens. Detailed studies around the properties of these antibodies have shown that they are of low affinity, germ-line or near germ-line in configuration and predominantly IgM, but some also are IgG or IgA1,2,3. Functional studies have revealed that this broad anti-bacterial activity of normal serum is largely due to polyreactive antibodies which bind to a variety of bacteria, fix match and enhance phagocytosis4,5,6. In addition polyreactive antibodies can bind to cells made apoptotic by UV light or HIV contamination. Upon binding to the apoptotic cells, the polyreactive antibodies also can fix match and participate in the clearance or phagocytosis of these cells by macrophages7,8. Thus, polyreactive antibodies protect the host against both foreign invaders and its own damaged apoptotic cells before the onset of the adaptive immune response. Still other studies suggest that polyreactive antibodies may contribute to the enhancement of immune tolerance by transporting peripheral antigens to the thymus9and under some circumstances polyreactive antibodies are thought to have therapeutic potential10,11. The B cells that make these polyreactive antibodies are found in the peritoneal and pleural cavities Il6 as well as peripheral lymph nodes. In the cord blood of newborns about 50% of the B cells make polyreactive antibodies and in the peripheral blood circulation of adults about 15 to 20% of the B cells make polyreactive antibodies12,13,14. In normal serum, it is estimated that there are millions of polyreactive antibodies, many of which show different binding patterns and affinities when evaluated with a large panel of antigens. Thus, quantification of polyreactive antibodies that react with hundreds or thousands of different antigens requires a different approach than the quantification of antibodies that react with a single antigen (i.e., antigen-specific). To develop an assay for measuring polyreactive antibodies, we chose a synthetic molecule , dinitrophenol (DNP), which is not present in the environment and to which the host would not be exposed. Therefore, antibodies in the serum that react with DNP would almost certainly be polyreactive. Using two fold serial dilutions of serum, as recently described15, we have been able to determine the end-point or titer of polyreactive antibodies in a variety of normal and pathophysiologic says. Unanswered, however, is what actually triggers the secretion of polyreactive antibodies that are present in normal sera. Because of the low BEC HCl affinity of polyreactive antibodies, it is unlikely that activation by one or more of the serum antigens which might bind to polyreactive B cell receptors, would provide a strong enough signal to result in the secretion of polyreactive antibodies. Rather, we hypothesized that this activation of Toll-Like Receptors (TLRs) on B lymphocytes, by their respective agonists, would provide strong enough stimuli to trigger the secretion of polyreactive antibodies into serum. The current study was initiated to test this possibility. == Results == == The TLR4 agonist, lipopolysaccharide (LPS), stimulates the secretion of polyreactive antibodies into the peripheral blood circulation == To determine the effect of TLRs around the secretion of polyreactive antibodies, mice were injected with LPS, a potent TLR4 ligand. As seen inFig. 1A, the titer of polyreactive IgM antibodies in sera increased about 40 fold, from 2,100 to 85,000, at 96 hours after the injection of LPS. Proof that this increase was the result of TLR4 activation came from performing the same experiment in TLR4 null mice. As seen inFig. 1B, there was only a 2 fold increase in the titer of BEC HCl polyreactive antibodies. Similarly, the injection of LPS in mice deficient in MyD88, which is one of the adaptor proteins required for the downstream activation of TLR signaling, also resulted in only a two fold increase in the titer of polyreactive antibodies at 96 hours (Fig. 1C). Comparable findings BEC HCl were observed by measuring polyreactive IgG (Suppl. Fig. 1). Since it is well known that activation of TLR4 with LPS results in a substantial increase.