Currently, treatment of sepsis is ineffective and few therapeutic innovations have been developed to improve it [1, 2]. Since 1972, stemming from the quasi-intuitive ideas of Thomas Lewis , the concept that our immune response to infection is the sustaining foundation for sepsis has been widely accepted. An increased understanding of sepsis pathophysiology has provided stronger support for this hypothesis . During the second half of the 1990 s, two coinciding facts led to new perspectives on a different approach to sepsis. First, the discovery of toll-like receptors (TLR) [5, 6] initiated our present comprehension of how the host recognizes pathogen molecular patterns, how the innate response is initiated, and how the acquired immune response is organized . Second, in 1996, Bernd Echtenacher et al. determined the essential role of mast cells in the innate immune response using a model of peritoneal sepsis in mast cell-deficient mice . Additional work from other groups (reviewed in 9) elevated these cells, previously considered mere effectors, to the category of sentinels of the innate immune system and organizers of the adaptive immune response. In fact, their permanent location in sites likely to suffer invasion by pathogens - skin, paranasal sinuses, lungs, and intestinal mucosa - places them in a privileged position in terms of detection and subsequent organization of the immune response. Innumerable possibilities for modulation of the inflammatory response appeared after a better understanding of the role played by TLRs was gained, having as targets their stimulatory and inhibitory signaling pathways . The accumulation of evidence defining mast cells as fundamental in the immune response to sepsis [11–13] opens up new perspectives on the mechanisms of immunomodulation by this type of cell .
Interaction between responses to infectious and allergic stimuli has been suggested on several levels, raising the possibility of a common pathway. Genetics-based studies have revealed the association of polymorphisms in the myosin light chain kinase (MYLK) gene with increased risk of sepsis and acute pulmonary injury. This same gene is involved in other inflammatory pathologies, including bronchial asthma . Epidemiological data have demonstrated the effect of exposure to TLR agonists on the incidence of allergic phenomena . Experimental studies in human mast cell cultures have shown an interaction between FcεR1 receptors and TLR2 . In bone marrow derived-mast cells (BMMC), we have previously demonstrated the synergic action of co-stimulation of FcεR1 and TLR in the production of inflammatory cytokines .
The endotoxin tolerance phenomenon was described more than 60 years ago , and it is characterized by hyporesponsiveness to endotoxin exposure, induced by prior exposure. Cross-tolerance, which is defined by tolerance of a different stimulus induced by endotoxin, was described later. In the context of TLR signaling, the tolerance phenomenon can be used as a tool for identification of signaling molecules that can attenuate the inflammatory response, revealing potential participants in immunomodulation. Mast cells are some of the first cells to have contact with invading pathogens; when activated, they release immunoregulatory cytokines that organize the inflammatory response. The release of TNFα and the recruitment of circulating leukocytes are essential elements of the immune response that are attributed to mast cells, and characteristically, mast cells are the only type of cell that can store pre-formed TNFα and release it when activated . Few studies have addressed endotoxin tolerance in mast cells, likely due to the fact that its primary role in response to infection has been defined so recently. In other cell types, where endotoxin tolerance have been more thoroughly evaluated, the proteins suppressors of cytokine signaling (SOCS) are involved in tolerance phenomena as negative regulators of the pro-inflammatory response, via the TLR4-NFκB pathway .
We tested the hypothesis that mast cells display the phenomenon of direct tolerance to endotoxins after prestimulation of BMMC with LPS, and that this could induce cross-tolerance for stimulation with FcεR1 and TLR2 agonists. The release of TNFα and IL-6 was measured, and the same experiment was conducted in BMMC obtained from TLR4-/- knock-out (KO) mice to determine the role of TLR4 in the induction of cross-tolerance and in the potentiation of this response by co-stimulation. Additionally, we evaluated the effects of LPS prestimulation on phosphorylation of the mitogen-activated protein kinase, p38, and the transcription factor NFκB, as well as the expression of the SOCS-1 and -3 proteins, signaling molecules involved in cytokine production.