Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane.

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.

Differential effect of soluble fibrinogen as a neutrophil activator.

A fundamental paradigm involved in acute inflammatory responses to invading pathogens and tissue damage is the migration of specific leukocyte populations to the affected tissues to mount an initial innate response to the aggression. The recruitment of polymorphonuclear neutrophils (PMNs) from the blood is a central event in this respect. The aim of this study was to understand whether fibrinogen is able to modulate the pattern of neutrophil activation and thus contribute to neutrophil recruitment. We demonstrated that fibrinogen induces free radical production by neutrophils without modifying the activation status of Mac-1 (αMß2, CD11b/CD18), the previously identified neutrophil receptor for fibrinogen. This data indicates that fibrinogen must have an additional different binding site in the neutrophil membrane. Importantly, we propose that as Mac-1 activation was not affected by the binding of fibrinogen, activated neutrophils can further maintain their ability to marginate, roll and adhere to the endothelial walls.

Fibrinogen modulates leukocyte recruitment in vivo during the acute inflammatory response.

Besides playing an important role in blood hemostases, fibrinogen also regulates leukocyte function in inflammation. Our previous in vitro studies showed that the adhesive behaviour of the neutrophil is modulated by soluble fibrinogen when present at a physiological concentration. This led us to propose that this plasma glycoprotein might further influence leukocyte recruitment in vivo and thus contribute to the inflammatory response. To address this in vivo, leukocyte recruitment was here investigated under acute inflammatory conditions in the absence of soluble fibrinogen in the blood circulation. For such, intravital microscopy on mesentery post-capillary venules was performed on homozygous fibrinogen α chain-deficient mice ((α-/-) mice). Acute inflammatory states were induced by perfusing platelet activating factor (PAF) over the exposed tissue. As control animals, two groups of mice expressing soluble fibrinogen in circulation were used, namely, C57BL/6 wild type animals and heterozygous fibrinogen α chain-deficient mice ((α+/-) mice). Under acute inflammatory conditions, an abnormal pattern of recruitment was observed for leukocytes in homozygous (α-/-) mice in comparison to both control groups. In fact, the former exhibited a significantly decreased number of rolling leukocytes that nevertheless, migrated with increased rolling velocities when compared to leukocytes from control animals. Consistently, homozygous mice further displayed a diminished number of adherent leukocytes than the other groups. Altogether our observations led us to conclude that leukocyte recruitment in homozygous (α-/-) mice is compromised what strongly suggests a role for soluble fibrinogen in leukocyte recruitment in inflammation.

[Vasoactive endothelial factors]. / Factores endoteliais vasoactivos.

Endothelium-derived vasoactive factors are produced by the endothelium activated by effective stimulus, and with paracrine regulatory activity of the tone/proliferation of the vascular smooth muscle and platelet function. They are divided in two groups: endothelium-derived relaxing and contracting factors. Among the endothelium-derived relaxing factors, PG I2, EDRF (NO or other nitrous compound) and EDHF (still unidentified) have been considered Synthetized by the endothelium after stimulation by plasmatic, platelet-derived and endothelium-derived substances and mechanisms, towards the vascular smooth muscle (myorelaxing/cytostatic) and the platelets (antiaggregation). The endothelium-derived contracting factors include the EDCF1 (endothelins, 21 amino acids peptides), EDCF2 (O2-) and TxA2. Its production, induced by stimulus similar to those for relaxing factors, promotes constriction/mitogenesis of the vascular smooth muscle and platelet aggregation. Probably, endothelin-1 has indirect actions over hormonal mechanisms of cardiovascular and renal regulation. The vascular system establishes a tight regulation over the production of these endothelium-derived vasoactive factors. Its loss (usually due to alteration of endothelial responsiveness to stimulation) allows local or generalized modifications of the vascular tone. These can depend on hypertension, atherosclerosis, ischemia-reperfusion lesion, diabetes, inflammation and situations of farmacotoxicity (all developing vasoconstriction/vasospasm) or by septicemia (leading to vasodilation). This disregulation is also involved in the pathogenesis of hypertension, atherosclerosis and ischemia-reperfusion. The vascular tone regulation by endothelium also leads to systemic consequences. Essentially by decreasing cardiac, cerebral and renal blood flow it implies morphologic and functional modifications of these organs.

An in vitro study on the modulation of the neutrophil adhesive behavior by soluble fibrinogen.

Fibrinogen constitutes an important plasma glycoprotein involved in hemostasis and in inflammation. Previously, we have shown that at physiological concentrations, soluble fibrinogen is able to modulate the pattern of neutrophil activation. This led us to propose that under these conditions, fibrinogen could as well interfere with the adhesive behaviour of circulating neutrophils which is of utmost importance in their recruitment to the vascular wall during inflammatory processes. To address our working hypothesis, in vitro adhesion assays were here performed in a flow chamber by using primary cultures of human umbilical vein endothelial cells (HUVEC) and neutrophils isolated from peripheral venous blood of healthy human donors. In the presence of a physiological concentration of soluble fibrinogen (300 mg/dL), we observed that despite the number of neutrophils rolling on an activated endothelium was not affected, their rolling velocity was increased in comparison to that of non-activated neutrophils. Consequently as expected, the number of fibrinogen-treated neutrophils adhering to activated HUVEC monolayers was significantly diminished. Overall, we have here demonstrated that at least in vitro, soluble fibrinogen under physiological concentrations is able to modulate the interaction of neutrophils with the vascular endothelium. In vivo studies will enable us in the future to study the physiological relevance of these findings and further to understand the mechanisms underlying this function.

Neutrophils in rheumatoid arthritis: More than simple final effectors.

Rheumatoid arthritis is the most common inflammatory joint disease. The etiopathogenesis of this condition has been classically explained by a T cell-driven process. However, recent studies have highlighted the possible contribution of neutrophils for the early phases of RA physiopathology. These cells are phagocytic leukocytes that play crucial roles in the acute defense against pathogens while modulating the function of other immune cells and contributing to the perpetuation of an initial inflammatory response. The herein article reviews recent progresses in the understanding of the immunopathology of RA with a special emphasis on the role of neutrophils.

Neutrophils: warriors and commanders in immune mediated inflammatory diseases.

Neutrophils are critical effector cells in the immune system. They not only play crucial roles in pathogenic defense but are also able to modulate the function of other immune cells and consequently contribute to the immune response fate. The herein review is focused in neutrophil biology in a general perspective and its contribution to the course of immune mediated inflammatory diseases.