Inhibit Leukocyte Function Research Articles

Staphylococcal SSL5-induced platelet microparticles provoke proinflammatory responses via the CD40/TRAF6/NFκB signalling pathway in monocytes.

Pathogens-induced platelet activation contributes to inflammation in cardiovascular diseases, but underlying mechanisms remain elusive. Staphylococcal superantigen-like protein 5 (SSL5) is a known activator of platelets. Here we examined whether SSL5 is implicated in Staphylococcus aureus (S. aureus)-induced inflammation and potential mechanisms involved. As expected, we show that SSL5 activates human platelets and induces generation of platelet microparticles (PMPs). Flow cytometry and scanning electron microscopy studies demonstrate that SSL5-induced PMPs (SSL5-PMPs) bind to monocytes, causing aggregate formation. In addition, SSL5-PMPs provoke monocyte expression and release of inflammatory mediators, including interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) in a dose- and time-dependent manner. SSL5-PMPs also enhance MCP-1-induced monocyte migration. Blockade of CD40 and CD40 ligand (CD40L) interactions with neutralising antibodies significantly reduce monocyte release of inflammatory mediators and migration induced by SSL5-PMPs. SiRNA-mediated silencing of CD40 or TNF receptor (TNFR)-associated factor 6 (TRAF6) gene largely abrogates phosphorylation and nuclear translocation of NFκB (p65). In conclusion, SSL5 provokes the release of inflammatory mediators in monocytes, at least in part, via PMPs-mediated activation of the CD40/TRAF6/NFκB signalling pathway, though it normally inhibits leukocyte function. Our findings thus reveal a novel mechanism by which S. aureus induces inflammation.

Abstract 413: Designed Ankyrin Repeat Proteins Against Activation Specific Binding Sites of The Leukocyte Integrin αmβ2: Novel Strategies in Vascular Inflammation

Introduction: Vascular inflammation is the underlying condition of several cardiovascular diseases and is mainly mediated by activated leukocytes. The leukocyte integrin αMβ2 with its activation specific epitope (I domain) is strongly involved in leukocyte adhesion to endothelial cells and thus represents an interesting therapeutic target. Designed Ankyrin Repeat Proteins (DARPins) are a novel class of linear, thermostable, highly specific recombinant binding proteins that overcome several limitations of immunoglobulins. Hypothesis: DARPins selected against the mouse I domain (mId) of αMβ2 bind specifically to activated leukocytes and can be used as a novel diagnostic tool as well as a therapeutic, anti-inflammatory agent. Methods: Using phage display, binding proteins were selected against recombinant I domain. Specific binding behavior to only activated leukocytes was assessed in FACS. Docking studies were used to define specific interaction sites of selected DARPins with the I domain. Therapeutic, anti-inflammatory effects of anti-mId DARPins was assessed in a sepsis mouse model. Results: DARPins selected against the I domain bind in FACS specifically to activated monocytes (activated vs. non-activated 61±4 % vs. 19±6 %, p<0.05). Docking studies revealed amino acid positions responsible for the specific binding behavior. Mutagenesis of these residues showed significantly reduced binding of the mutated DARPin using FACS analysis (anti-mId DARPin vs. mutated anti-mId DARPin 61±4 vs. 29±7, p<0.05) proving that binding of the wild type DARPin to its target is specific. Furthermore, anti- I domain DARPins showed anti-inflammatory effects in a mouse sepsis model (peritoneal cells: anti-mId DARPin vs control: 2,049±189 103/ml vs. 3,382±213 103/ml, p<0.01). Conclusions: DARPins selected against the I domain of αMβ2 bind specifically to activated leukocytes and inhibit leukocyte function as a new class of anti-inflammatory agents under in vivo conditions.

Innate Immune Function and Mortality in Critically Ill Children With Influenza: A Multicenter Study

MW Hall, SM Geyer, CY Guo; Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network PICFlu Study Investigators. Crit Care Med. 2013;41(1):224–236 The influenza virus can inhibit leukocyte function in vitro and in vivo, and is associated with severe secondary bacterial infections. Immunoparalysis is the state marked by reduction in the ability to produce tumor necrosis factor (TNF)-α in response to lipopolysaccharide (LPS). This study sought to test the hypothesis that mortality from influenza in critically ill children and young adults is associated with both hypercytokinemia and innate immune suppression. Patients <18 years old, who were admitted to 1 of the 15 PICUs that belong to the Pediatric Acute Lung …

Blocking LFA-1 Activation with Lovastatin Prevents Graft-versus-Host Disease in Mouse Bone Marrow Transplantation

Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function-associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low-affinity state to the high-affinity state on LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In the present study, we report that lovastatin blocked mouse T cell adhesion, proliferation, and cytokine production in vitro. Furthermore, blocking LFA-1 in the low-affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes from homing to lymph nodes and Peyer's patches during the GVHD initiation phase and after donor lymphocyte infusion (DLI) after the establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD.

Differential Roles of Galectin-1 and Galectin-3 in Regulating Leukocyte Viability and Cytokine Secretion

Galectin-1 (Gal-1) and galectin-3 (Gal-3) exhibit profound but unique immunomodulatory activities in animals but their molecular mechanisms are incompletely understood. Early studies suggested that Gal-1 inhibits leukocyte function by inducing apoptotic cell death and removal, but recent studies show that some galectins induce exposure of the common death signal phosphatidylserine (PS) independently of apoptosis. In this study, we report that Gal-3, but not Gal-1, induces both PS exposure and apoptosis in primary activated human T cells, whereas both Gal-1 and Gal-3 induce PS exposure in neutrophils in the absence of cell death. Gal-1 and Gal-3 bind differently to the surfaces of T cells and only Gal-3 mobilizes intracellular Ca2+ in these cells, although Gal-1 and Gal-3 bind their respective T cell ligands with similar affinities. Although Gal-1 does not alter T cell viability, it induces IL-10 production and attenuates IFN-gamma production in activated T cells, suggesting a mechanism for Gal-1-mediated immunosuppression in vivo. These studies demonstrate that Gal-1 and Gal-3 induce differential responses in T cells and neutrophils, and identify the first factor, Gal-3, capable of inducing PS exposure with or without accompanying apoptosis in different leukocytes, thus providing a possible mechanism for galectin-mediated immunomodulation in vivo.

Modulation of adhesion molecule expression on endothelial cells: to be or not to be?

Modulation of adhesion molecule expression on endothelial cells: to be or not to be?

Therapeutic modification of leukocyte homing to the skin

This review article provides an excellent introduction to the immunopathology of skin. The authors describe the mechanisms that trigger leukocyte migration into inflamed skin, highlighting the role of the cutaneous lymphocyte antigen (CLA) in this process, and after summarize the major T-cell mediated skin disorders. Their final conclusion is that targeting the processes that regulate the homing of T-lymphocytes to the skin is likely to be a major advance for the treatment of these inflammatory skin diseases.

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.

Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat.

Agents that inhibit leukocyte adhesion including intercellular adhesion molecule-1 antibodies (anti-ICAM-1) have shown beneficial effects in experimental central nervous system (CNS) ischemia. Doxycycline inhibits leukocyte function in vitro by binding divalent cations and reduces spinal cord reperfusion injury. The authors used a clinically relevant model of focal CNS reperfusion injury to test whether treatment with doxycycline would reduce cerebral ischemic damage and improve functional outcome. Reversible middle cerebral artery occlusion was produced in adult Sprague-Dawley rats by advancing a filament into the internal carotid artery for 2 h. Animals received either i.p. doxycycline (10 mg/kg) (N = 13) or saline (N = 11) 30 min before ischemia, followed by 10 mg/kg every 8 h x 6. Both functional assessment (5 point neurologic scale) and infarct volume was evaluated at 48 h. Functional efficacy: doxycycline 0.5 +/- 0.2 (mean +/- SE) vs control 1.3 +/- 0.3 (p = 0.03). Infarct volume: doxycycline 56 +/- 18 mm3 vs control 158 +/- 44 mm3 (p = 0.03); This protective effect supports the role of doxycycline in reducing CNS reperfusion injury.

In VitroStudies on the Effect of Dialysis Solutions on Peritoneal Leukocytes

Within the limitations of the various experimental protocols there appears to be agreement in the literature that unused dialysis fluids, at least when studied in vitro, adversely affect multiple leukocyte functions. The effects of dialysis fluids on leukocytes that have been reported to date include: 1. Decreased cell viability of PMNs, PM phis, PBMCs, and lymphocytes; 2. Inhibited phagocytosis and bacterial killing of various microorganisms by PM phis, PMNs, and peripheral blood leukocytes; 3. Reduced secretion of leukotrienes (LTB4, LTC4) from peritoneal and peripheral blood PMNs and PBMCs; 4. Reduced secretion of prostaglandins (PGE2, TXB2 and 6-keto-PGF1 alpha) from PM phi; 5. Decreased production of many cytokines including TNF alpha, IL-8, and IL-6 in PM phis and PBMCs. In addition, several studies targeting the potential mechanisms by which dialysis solutions inhibit leukocyte function identified the initial low pH of the fluids in combination with their lactate content as being of primary relevance, since they may lead to a rapid intracellular acidification of leukocytes. Moreover, some studies indicated the importance of fluid hyperosmolarity and excessive glucose concentrations. These results are indirectly supported by recent in vitro investigations of alternative fluids, which showed improved leukocyte function following exposure to solutions with neutral pH, bicarbonate buffer instead of lactate, or normal osmolarity due to use of an alternative osmotic agent (e.g., glucose polymer). In conclusion, the evidence obtained during in vitro experimentation suggests that current dialysis fluids are, indeed, not biocompatible. However, whether this also bears physiological relevance in vivo remains to be established in controlled clinical trials comparing conventional fluids to alternative solutions with improved biocompatibility. With regard to the future development of in vitro models for biocompatibility assessment, the following guidelines are suggested: 1. Cell functional parameters should be studied in more than one cell population; 2. Depending on which fluid aspect is under investigation, short or even very short exposure times should be used (e.g., < 30 min for pH/buffer studies; < 4 hours for osmolality/osmotic agent studies); 3. In case the parameter/readout of interest requires longer study periods than indicated above (e.g., studies of cytokine induction or surface receptor expression), preincubation/recovery models should be preferred over coincubation experiments.

Reduction of central nervous system reperfusion injury in rabbits using doxycycline treatment.

Activated leukocytes appear to potentiate central nervous system reperfusion injury, and agents that block leukocyte adhesion have shown neuroprotective efficacy in experimental models. Doxycycline, a tetracycline antibiotic, inhibits leukocyte function in vitro, presumably through divalent cation binding. We used a model of focal central nervous system reperfusion injury to determine the efficacy of doxycycline treatment in preserving neurological function. Rabbits randomly received 10 mg/kg i.v. doxycycline 30 minutes before ischemia (pretreatment group) or 45 minutes after ischemia (posttreatment group) or received phosphate-buffered saline vehicle (control group) followed by 10 mg/kg q 8 hours times two. The average length of reversible spinal cord ischemia required to produce paraplegia (P50) at 18 hours was calculated for each group. For the control group (n = 13), the P50 was 22.8 +/- 2.2 minutes; for the pretreatment group (n = 14), 35.5 +/- 2.4 minutes (P < .01; t = 3.8); and for the posttreatment group (n = 13), 31.4 +/- 4.2 minutes (not significant; t = 1.6). Doxycycline also attenuated postischemic decreases in vivo leukocyte counts and inhibited in vitro leukocyte adhesion. Therapeutic doxycycline levels at 24 hours were confirmed in the plasma and spinal cord. This significant protective effect suggests that doxycycline, a safe and readily available agent, may play a role in reducing clinical central nervous system reperfusion injury.

Soluble intercellular adhesion molecule 1-immunoglobulin G1 immunoadhesin mediates phagocytosis of malaria-infected erythrocytes.

We describe an immunoadhesin molecule containing intercellular adhesion molecule 1 (ICAM-1) molecularly fused to hinge and CH2 and CH3 domains of the human immunoglobulin G1 H chain that binds Plasmodium falciparum-infected erythrocytes. This receptor-based immunoadhesin is an effective and specific inhibitor of P. falciparum-infected erythrocyte adhesion to ICAM-1-bearing surfaces, but does not inhibit leukocyte function antigen 1 (LFA-1) interaction with ICAM-1. Furthermore, the immunoadhesin promotes phagocytosis and destruction of parasitized erythrocytes by human monocytes. Each of these modes of action has potential for the therapy of malaria.

Effect of oral N-acetylcysteine administration on human blood neutrophil and monocyte function.

N-acetylcysteine (NAC) is known to be a scavenger of free oxygen radicals, and recent in vitro studies have demonstrated that it is also able to inhibit leukocyte function. The clinical significance of these effects is, however, not known. In this study we have measured the effect on human blood neutrophil and monocyte function of a single 400 mg dose of NAC administered orally. Administration of NAC to ten healthy volunteers resulted in significant reduction of neutrophil chemiluminescence response following activation by opsonized zymosan as compared to four non-treated persons acting as controls. No effect was observed on the chemotaxis of either cell type or on monocyte chemiluminescence response. These findings suggest that NAC may be beneficial in clinical conditions like cystic fibrosis, where tissue damage may be a consequence of the effects of increased release of toxic oxygen radicals and proteolytic enzymes.