Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma.

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC).

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αßγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8ß contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8ß, rotation of the regulatory segment is linked to an opening of the central ß-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the ß-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.

The chemokine CCL18 causes maturation of cultured monocytes to macrophages in the M2 spectrum.

The observation that human monocytes cultured in the presence of the chemokine CCL18 showed increased survival, led us to profile cytokine expression in CCL18-stimulated versus control cultures. CCL18 caused significantly increased expression of chemokines (CXCL8, CCL2, CCL3 and CCL22), interleukin-10 (IL-10) and platelet-derived growth factor, but no up-regulation of M1 cytokines IL-1ß or IL-12. CCL18-stimulated monocytes matured into cells with morphological resemblance to IL-4-stimulated macrophages, and expressed the monocyte marker CD14 as well the M2 macrophage markers CD206 and 15-lipoxygenase, but no mature dendritic cell markers (CD80, CD83 or CD86). Functionally, CCL18-stimulated macrophages showed a high capacity for unspecific phagocytosis and for pinocytosis, which was not associated with an oxidative burst. These findings suggest that CCL18-activated macrophages stand at the cross-roads between inflammation and its resolution. The chemokines that are produced in response to CCL18 are angiogenic and attract various leucocyte populations, which sustain inflammation. However, the capacity of these cells to remove cellular debris without causing oxidative damage and the production of the anti-inflammatory IL-10 will initiate termination of the inflammatory response. In summary, CCL18 induces an M2 spectrum macrophage phenotype in the absence of IL-4.

Expression of soluble proteins in Escherichia coli by linkage with the acidic propiece of eosinophil major basic protein.

An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP. It was reasoned that constructs of this acidic domain with small heterologous cationic proteins expressed in E. coli could result in soluble expression while preventing trafficking and packaging into insoluble inclusion bodies. This has been demonstrated using four examples: complement C5a, CCL18, fibroblast growth factor-ß, and leukemia inhibitory factor, whose isoelectric points range from 8.93 to 9.59. Further general applicability of this technique has been shown by using two different expression systems, one which encodes an amino-terminal oligo-histidine leash, and another that codes for an amino-terminal glutathione-S-transferase. Thus the utility of coupling MAP to cationic polypeptides for the purpose of soluble heterologous protein expression in E. coli has been demonstrated.

C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation.

Mesenchymal stem cells (MSCs) have a great potential for tissue repair, especially if they can be delivered efficiently to sites of tissue injury. Since complement activation occurs whenever there is tissue damage, the effects of the complement activation products C3a and C5a on MSCs were examined. Both C3a and C5a were chemoattractants for human bone marrow-derived MSCs, which expressed both the C3a receptor (C3aR) and the C5a receptor (C5aR; CD88) on the cell surface. Specific C3aR and C5aR inhibitors blocked the chemotactic response, as did pertussis toxin, indicating that the response was mediated by the known anaphylatoxin receptors in a G(i) activation-dependent fashion. While C5a causes strong and prolonged activation of various signaling pathways in many different cell types, the response observed with C3a is generally transient and weak. However, we show herein that in MSCs both C3a and C5a caused prolonged and robust ERK1/2 and Akt phosphorylation. Phospho-ERK1/2 was translocated to the nucleus in both C3a and C5a-stimulated MSCs, which was associated with subsequent phosphorylation of the transcription factor Elk, which could not be detected in other cell types stimulated with C3a. More surprisingly, the C3aR itself was translocated to the nucleus in C3a-stimulated MSCs, especially at low cell densities. Since nuclear activation/translocation of G protein-coupled receptors has been shown to induce long-term effects, this novel observation implies that C3a exerts far-reaching consequences on MSC biology. These results suggest that the anaphylatoxins C3a and C5a present in injured tissues contribute to the recruitment of MSCs and regulation of their behavior.

Akt plays an important role in breast cancer cell chemotaxis to CXCL12.

The chemokine receptor CXCR4 is functionally expressed on the cell surface of various cancer cells, and plays a role in cell proliferation and migration of these cells. Specifically, in breast cancer cells the CXCR4/CXCL12 axis has been implicated in cell migration in vitro and in metastasis in vivo, but the underlying signaling mechanisms are incompletely understood. The xenograft-derived MDA-MB-231 breast cancer cell line (231mfp), which was shown previously to grow more aggressively than the parent cells, showed increased CXCR4 expression at the mRNA, total protein and cell surface expression level. This correlated with an enhanced response to CXCL12, specifically in augmented and prolonged Akt activation in a G(i), Src family kinase and PI-3 kinase dependent fashion. 231mfp cells migrated towards CXCL12--in contrast to the parent cell line--and this chemotaxis was blocked by inhibition of G(i), Src family kinases, PI-3 kinase and interestingly, Akt itself, as could be shown with two pharmacological inhibitors, a dominant negative Akt construct and with Akt shRNA. Collectively, we have demonstrated that prolonged Akt activation is an important signaling pathway for breast cancer cells expressing CXCR4 and is necessary for CXCL12-dependent cell migration.

The role of the complement anaphylatoxins in the recruitment of eosinophils.

Eosinophils are blood and tissue immune cells that participate in a diverse range of activities normally beneficial for the host defense, but in circumstances of untoward inflammatory conditions these cells can be responsible for pathological responses. Accordingly the transit of eosinophils from the blood to tissues is a subject of considerable importance in immunology. In this article we review how the complement anaphylatoxins, C3a and C5a bring about eosinophil extravasation. These mediators do not merely provide a chemotactic or haptotactic gradient but are responsible for orchestrating innumerable responses by other cells types, including of endothelial cells, mast cells, and basophils in order to create an environment that is conducive for eosinophil infiltration. C5a has the capacity to prime the endothelium directly to present P-selectin, and C5a stimulated generation of eosinophil hydrogen peroxide and other oxidants can cause additional upregulation of endothelial P-selectin and ICAM-1. Moreover, the anaphylatoxins have the ability to recruit mast cells and basophils and can stimulate these cells to release IL-4 and IL-13, which by augmenting endothelial VCAM-1, convey some selectivity for eosinophils. The anaphylatoxins also have the capability to evoke the release and activation of eosinophil MMP-9, which is employed by this cell type to digest its way past the subendothelial matrix. Finally, because C3a and C5a can stimulate the generation of nitric oxide along with the secretion of histamine and LTC4 from several cell types, the anaphylatoxins can bring about an increase in vascular permeability that facilitates eosinophil accumulation at sites of allergic inflammation.

Magnetic bead based assays for complement component C5.

Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and ß-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only ß-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7.

C5a mediates secretion and activation of matrix metalloproteinase 9 from human eosinophils and neutrophils.

Matrix metalloproteinase 9 (MMP-9) is a crucial proteinase, utilized by both eosinophils and neutrophils, that mediates transmigration through extracellular basement membranes. We have found that neutralization of MMP-9 by a monoclonal antibody or a chemical inhibitor blocked C5a dependent chemotaxis of these granulocytes in vitro. The levels of MMP-9 secreted by the action of C5a from eosinophils were about 50-fold lower than those from neutrophils, consistent with results from confocal microscopy, where the density of MMP-9 containing granules was fewer within eosinophils than in neutrophils. Zymography indicated gelatin degrading activity of the molecular size of pro MMP-9 in supernatants from eosinophils and neutrophils stimulated by C5a, with no evidence of proteolytic activation. Instead MMP-9 activation appeared oxidative, since inhibition of NADPH oxidase and nitric oxide synthase by DPI or L-NIL abrogated C5a-mediated chemotaxis through basement membranes. In keeping with this mode of activation, C5a, known as an agent of superoxide generation, was also found to induce secretion of nitric oxide from human eosinophils and rat granulocytes and monocytes. In conclusion C5a is an important mediator that brings about secretion and oxidative activation of MMP-9, a requisite protease for transmigration, from both eosinophils and neutrophils.

Complement activation in the context of stem cells and tissue repair.

The complement pathway is best known for its role in immune surveillance and inflammation. However, its ability of opsonizing and removing not only pathogens, but also necrotic and apoptotic cells, is a phylogenetically ancient means of initiating tissue repair. The means and mechanisms of complement-mediated tissue repair are discussed in this review. There is increasing evidence that complement activation contributes to tissue repair at several levels. These range from the chemo-attraction of stem and progenitor cells to areas of complement activation, to increased survival of various cell types in the presence of split products of complement, and to the production of trophic factors by cells activated by the anaphylatoxins C3a and C5a. This repair aspect of complement biology has not found sufficient appreciation until recently. The following will examine this aspect of complement biology with an emphasis on the anaphylatoxins C3a and C5a.

Hyaluronan in the healthy and malignant hematopoietic microenvironment.

The fate of both endogenous and transplanted stem cells is dependent on the functional status of the regulatory local microenvironment, which is compromised by disease and therapeutic intervention. The glycosaminoglycan hyaluronan (HA) is a critical component of the hematopoietic microenvironment. We summarize recent advances in our understanding of the role of HA in regulating mesenchymal stem cells, osteoblasts, fibroblasts, macrophages, and endothelium in bone marrow (BM) and their crosstalk within the hematopoietic microenvironment. HA not only determines the volume, hydration, and microfluidics of the BM interstitial space, but also, via interactions with specific receptors, regulates multiple cell functions including differentiation, migration, and production of regulatory factors. The effects of HA are dependent on the polymer size and are influenced by the formation of complexes with other molecules. In healthy BM, HA synthases and hyaluronidases form a molecular network that maintains extracellular HA levels within a discrete physiological window, but HA homeostasis is often perturbed in pathological conditions, including hematological malignancies. Recent studies have suggested that HA synthases may have functions beyond HA production and contribute to the intracellular regulatory machinery. We discuss a possible role for HA synthases, intracellular and extracellular HA in the malignant BM microenvironment, and resistance to therapy.

Mesenchymal stem cells and their microenvironment.

Mesenchymal stem cells (MSC) are multipotent stem cells that hold promise for an expanding list of therapeutic uses, not only due to their ability to differentiate into all connective tissues including bone, fat and cartilage, but additionally due to their trophic and anti-inflammatory effects which contribute to healing and tissue regeneration. Ongoing research is starting to illuminate important aspects of the microenvironmental niche, which supports MSC self-renewal. In this review, we summarize recent findings on cellular structures and molecular pathways that are involved in regulation of MSC self-renewal versus differentiation, and in retention of MSCs within the niche versus mobilization and recruitment to sites of injury. In addition, the contribution of MSCs to the structure and function of hematopoietic and cancerous niches is discussed.

Hyaluronan is required for generation of hematopoietic cells during differentiation of human embryonic stem cells.

Hyaluronan (HA) is an important component of the microenvironment in bone marrow, but its role in regulation of the development of hematopoietic cells is not well understood. To address the role of HA in regulation of human embryonic stem cell (hESC) differentiation into the hematopoietic lineage, we screened for genes encoding components of the HA pathway. Using gene arrays, we found that HA synthases and HA receptors are expressed in both undifferentiated and differentiating hESCs. Enzymatic degradation of HA resulted in decreased numbers of hematopoietic progenitors and lower numbers of CD45+ cells generated in HA-deprived embryoid bodies (EBs). In addition, deprivation of HA resulted in the inhibition of generation of CD31+ cells, stromal fibroblast-like cells and contracting myocytes in EBs. RT-PCR and immunocytochemistry revealed that HA deprivation did not influence the dynamics of OCT4 expression, but decreased the expression of BRY, an early mesoderm marker, and BMP2, a later mesoderm marker in differentiating EBs. In addition, the endoderm markers α-FP and SOX17 were decreased, whereas the expression of the ectoderm markers GFAP and FGF5 was higher in HA-deprived cultures. Our findings indicate that endogenously produced HA contributes to the network that regulates the differentiation of hESC and the generation of mesodermal lineage in general and hematopoietic cells specifically.

Hyaluronan inhibits postchemotherapy tumor regrowth in a colon carcinoma xenograft model.

Bone marrow hypoplasia and pancytopenia are among the most undesirable sequelae of chemotherapy for the treatment of cancer. We recently showed that hyaluronan (HA) facilitates hematopoietic recovery in tumor-free animals receiving chemotherapeutic agents. However, following a chemotherapeutic regimen in tumor-bearing animals, it is possible that residual tumor cells might respond to systemic injections of HA. Thus, in this study, we investigated the effect of HA on the regrowth of residual tumor cells following chemotherapy. As a model, we used the HCT-8 human colon carcinoma cell line, which expresses the HA receptor CD44, binds exogenous HA, and is susceptible to a chemotherapy protocol containing irinotecan and 5-fluorouracil in a human/mouse xenograft model. HCT-8 cells were implanted in severe combined immunodeficient mice, followed by irinotecan/5-fluorouracil treatment. After three rounds of chemotherapy, residual tumors were allowed to regrow in the presence or absence of HA. The dynamics of tumor regrowth in the group treated with HA was slower compared with the control group. By week 5 after tumor implantation, the difference in the size of regrown tumors was statistically significant and correlated with lower proliferation and higher apoptosis in HA-treated tumors as compared with controls. This finding provides evidence that HA treatment does not stimulate but delays the growth of residual cancer cells, which is an important parameter in establishing whether the use of HA can enhance current chemotherapeutic strategies.

Key players in the gene networks guiding ESCs toward mesoderm.

Embryonic stem cells (ESCs) offer a powerful in vitro model to study mechanisms implicated in cell fate decision. Developmental pathways by which pluripotent ESCs become committed to specific lineages are reflected in dynamic changes of signaling and transcriptional programs. However, the mechanisms that govern the regulatory intracellular networks underlying lineage fate decisions and differentiation programs remain poorly understood and differ significantly in different species. In this review we analyze the current understanding of the signaling mechanisms and transcriptional regulation of differentiation of murine and human ESCs into the mesoderm.

Alpha 7 subunit of nAChR regulates migration of human mesenchymal stem cells.

The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4. The receptor agonist nicotine caused calcium (Ca(++)) influx into hMSCs suggesting that the calcium ion channel alpha 7 homopolymer mediated this response. While high concentrations of nicotine (10(5)M) induced hMSC apoptosis, physiological concentrations (10(7)M) did not interfere with cell survival. At non-toxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited. The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs. These findings reveal an involvement of the non-neural cholinergic system in regulation of hMSC migration.

Regulation of CXCR4-mediated nuclear translocation of extracellular signal-related kinases 1 and 2.

Activation of the chemokine receptor CXCR4 by its agonist stromal cell-derived factor 1 (SDF-1) has been associated with cell migration and proliferation in many cell types, but the intracellular signaling cascades are incompletely defined. Here we show that CXCR4-dependent extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was mediated through the Ras/Raf pathway, as demonstrated with a dominant-negative Ras mutant and pharmacological inhibitors. The Src inhibitor 4-amino-5-methylphenyl-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and the Rho-kinase (ROCK) inhibitor N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y27632) also attenuated SDF-1-induced ERK1/2 phosphorylation. Involvement of Src could furthermore be demonstrated by Src phosphorylation and by the shortened ERK1/2 phosphorylation in SYF cells, which are Src/Yes/Fyn-deficient compared with Src-reconstituted Src(++) cells. Membrane translocation of RhoA could be detected similarly. A large portion of the SDF-1-mediated ERK phosphorylation was detected in the nucleus, as shown by Western blotting and confocal microscopy, and resulted in the phosphorylation of the transcription factor Elk. It is interesting that the nuclear accumulation of ERK1/2 and Elk phosphorylation was completely blocked by dominant-negative Rho, Y27632, PP1, and latrunculin B, indicating that the Rho/ROCK pathway, Src kinase, and the actin cytoskeleton were required in this process. In accordance, neither nuclear ERK phosphorylation nor Elk phosphorylation were observed in SYF cells stimulated with SDF-1 but were reconstituted in Src(++) cells. In summary, these results demonstrate that Src, Rho/ROCK, and an intact cytoskeleton contribute to overall ERK1/2 activation in SDF-1-stimulated cells and are indispensable for nuclear translocation of ERK1/2 and activation of transcription factors.

CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes.

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6-blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.

Adhesive interactions between human neural stem cells and inflamed human vascular endothelium are mediated by integrins.

Understanding the mechanisms by which stem cells home precisely to regions of injury or degeneration is of importance to both basic and applied regenerative medicine. Optimizing regenerative processes may depend on identifying the range of molecules that subserve stem cell trafficking. The "rolling" of extravasating cells on endothelium under conditions of physiological flow is the first essential step in the homing cascade and determines cell adhesion and transmigration. Using a laminar flow chamber to simulate physiological shear stress, we explored an aspect of this process by using human neural stem cells (hNSCs). We observed that the interactions between hNSCs and tumor necrosis factor-alpha (TNF-alpha)-stimulated human endothelium (simulating an inflamed milieu) are mediated by a subclass of integrins--alpha2, alpha6, and beta1, but not alpha4, alphav, or the chemokine-mediated pathway CXCR4-stromal cell-derived factor-1alpha--suggesting not only that the mechanisms mediating hNSC homing via the vasculature differ from the mechanisms mediating homing through parenchyma, but also that each step invokes a distinct pathway mediating a specialized function in the hNSC homing cascade. (TNF-alpha stimulation also upregulates vascular cell adhesion molecule-1 expression on the hNSCs themselves and increases NSC-endothelial interactions.) The selective use of integrin subgroups to mediate homing of cells of neuroectodermal origin may also be used to ensure that cells within the systemic circulation are delivered to the pathological region of a given organ to the exclusion of other, perhaps undesired, organs.

Nicotinic acetylcholine receptor-mediated stimulation of endothelial cells results in the arrest of haematopoietic progenitor cells on endothelium.

The function of endothelial cells that contribute to the regulation of haematopoietic stem/progenitor cells (HSPC) migration from peripheral blood into bone marrow can be influenced by extrinsic factors including nicotine. Therefore, the effect of nicotine on HSPC extravasation was studied. Using a parallel laminar flow chamber, we demonstrated an increase in the number of HSPC adhering to the nicotine-exposed endothelium under conditions of physiological shear stress in vitro. Nicotine-induced adhesion of HSPC was inhibited by mecamylamine, a non-selective nicotinic acetylcholine receptor (nAchR) antagonist. The enhanced adhesive interactions of HSPC with nicotine-exposed endothelial monolayers coincided with the nicotine-induced activation of endothelial cells. Nicotine induced fast cytoskeletal reorganization and formation of filopodia in endothelial cells through interaction with the non-neuronal nAchR expressed by these cells. In addition, nicotine treatment stimulated rapid phosphorylation of Erk1/2 and p-38 in endothelial cells. Finally, nicotine inhibited the stroma derived factor-1-mediated transendothelial migration of HSPC. Decreased migration of HSPC correlated with diminished matrix metalloproteinase-9 activity secreted by bone marrow cells and decreased expression of CD44 on the surface of endothelial cells. Overall, our data suggest that exposure to nicotine causes endothelial cell dysfunction and leads to the pathological arrest of HSPC on endothelium, interfering with their proper migration process.