Emerging Strategies to Enhance Homing and Engraftment of Hematopoietic Stem Cells.

Successful clinical outcomes from transplantation of hematopoietic stem cells (HSCs) depend upon efficient HSC homing to bone marrow (BM), subsequent engraftment, and, finally, BM repopulation. Homing of intravenously administered HSCs from peripheral blood (PB) through the circulation to the BM stem cell niches, which is the first critical step that precedes their engraftment, is enforced by chemotactic factors released in the BM microenvironment that chemoattract HSCs. These chemotactic factors include α-chemokine stromal-derived factor 1 (SDF-1), the bioactive phosphosphingolipids sphingosine-1-phosphate (S1P) and ceramid-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. Stem cells may also respond to a Ca(2+) or H(+) gradient by employing calcium- or proton-sensing receptors, respectively. In this review, we will present emerging strategies based on ex vivo manipulation of graft HSCs that are aimed at enhancing the responsiveness of HSCs to BM-secreted chemoattractants and/or promoting HSC adhesion and seeding efficiency in the BM microenvironment.

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3.

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.

Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury.

BACKGROUND: Lung injury manifest clinically as adult respiratory distress syndrome (ARDS) is a common cause of morbidity and mortality following acute pancreatitis (AP). Neutrophils play a critical role in the progression of AP to ARDS. C-x-C chemokines are potent neutrophil chemoattractants and activators and have been implicated in AP. AIMS: To evaluate the effect of blocking the C-x-C chemokine, cytokine induced neutrophil chemoattractant (CINC), in AP on pancreatic inflammation and the associated lung injury in rats. METHODS: AP was induced by hourly intraperitoneal injections of caerulein. Goat anti-CINC antibody was administered either before or after starting caerulein injections to evaluate the prophylactic and therapeutic effects, respectively. Severity of AP was determined by measuring plasma amylase, pancreatic water content, and pancreatic myeloperoxidase (MPO) activity as a measure of neutrophil sequestration in the pancreas. Lung injury was determined by measurement of pulmonary microvascular permeability and lung MPO activity. RESULTS: Treatment with anti-CINC antibody had little effect on caerulein induced pancreatic damage. However, it reduced the caerulein mediated increase in lung MPO activity as well as lung microvascular permeability when administered either prophylactically (lung MPO (fold increase over control): 1.53 (0.21) v. 3.30 (0.46), p<0.05; microvascular permeability (L/P%): 0.42 (0.07) v. 0.77 (0.11), p<0.05) or therapeutically (lung MPO (fold increase over control): 2.13 (0.10) v 4.42 (0.65), p<0.05; microvascular permeability (L/P%): 0.31 (0.05) v 0.79 (0.13), p<0.05). CONCLUSION: Treatment with anti-CINC antibody afforded significant protection against pancreatitis associated lung injury. These results suggest that CINC plays an important role in the systemic inflammatory response in AP.

The MultiScreen filtration system to measure chemoattractant-induced release of leukocyte granule enzymes by differentiated HL-60 cells or normal human monocytes.

A variety of chemoattractants initiate chemotaxis by selective binding to chemoattractant receptors (CARs), a subfamily of seven transmembranous G-protein coupled receptors (7TM-GPCRs) expressed in the leukocyte plasma membrane. Whatever the chemoattractant, signaling leading to chemotaxis involves several common biological steps which occur within seconds to minutes of CAR ligand binding. Though each step can be used to study the progress of the chemotaxis activation process. only certain biological events are suitable for monitoring chemotaxis signaling on large sample numbers as required for drug screening. An example of such is the release of granule enzymes by leukocytes in response to a CAR ligand. In this study, promyelocytic HL-60 cells were employed to set up a 96-well microplate methodology using filtration instead of centrifugation to collect the extracellular fluid together with the cell-released enzymes. Undifferentiated HL-60 cells were found not to respond to any of the CAR ligands. With various types of HL-60 cells which had differentiated along the neutrophilic or monocytic pathways, a large enzyme release was dose-dependently triggered by fMLF or C5a, but none of the tested CC or CXC chemokines. The highest responsiveness was found for neutrophilic HL-60 cells differentiated with dibutyryl cyclic AMP. With normal human monocytes (prepared from the blood of healthy donors by leukapheresis and elutriation), the granule enzyme release response was large to fMLF or C5a, substantial to MCP-1, low to RANTES or MIP-1alpha, but insignificant to Eotaxin, IL-8 and GROalpha. The method readily measures N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and elastase activities, and requires approximately five times fewer cells than classical methods, a very important feature when normal human cells are to be used in screening assays. The method was also adapted to large scale screening of antagonists such as cyclosporins A and H for fMLF-mediated signaling using HL-60 cells and monocytes, and truncated (9-76) MCP-1 for MCP-1-mediated signaling using monocytes.

Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists.

Basophils stimulated with IL-3 plus C5a selectively express IL-4 and IL-13 and continuously produce leukotrienes (LT) for hours. C5a combined with IL-5 or granulocyte-macrophage colony-stimulated factor was, however, much less effective in promoting cytokine expression and a late continuous phase of LTC4 production, possibly due to lower expression levels of their receptor alpha chains. Basophils also express several chemoattractant receptors, including high levels of C5a receptors, macrophage chemotactic protein (MCP) receptors (CCR2) and eotaxin receptors (CCR3), intermediate levels of CXCR1, CXCR2 and platelet-activating factor receptors, and lower levels of N-formyl-Met-Leu-Phe (fMLP) receptors. However, among the corresponding agonists, only C5a, fMLP and much more weakly MCP1, were found to induce cytokine expression and continuous LTC4 release, and only when combined with IL-3. CCR3, which is highly expressed on basophils and has been shown to mediate strong migratory but weak release responses, does not regulate cytokine expression. The weakly expressed fMLP receptor is an efficient activator of several cell functions including LTC4 formation, while CXCR2 hardly affects basophil function despite considerable expression. Thus, chemoattractant-receptors mediate different cellular responses unrelated to their expression levels.