Peptide fragments and structural analogues of chemokine MPC-1: synthesis and effect on the MPC-1-induced migration of monocytes

Fourteen fragments and structural analogues of chemokine MCP-1 were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of synthesized peptides on the MCP-1-stimulated migration of mononuclear cells was examined. Both in vitro stimulants and inhibitors of the monocyte migration were found among the peptides. A possible participation of the C-terminal part of the MCP-1 molecule in the inhibition of the MCP-1-stimulated cell migration was found for the first time. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.     

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.     

The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement

Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX(3)C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX(3)CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 A resolution. The CDF monomers form a dimer through an intermolecular beta-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX(3)C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivo is not well understood.     

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions

We have undertaken a study of the structure of the amino-terminal domain of the phytochrome polypeptide purified from Avena sativa L. Amino-acid sequencing was used to indentify arginine 52 as the precise location of a conformation-specific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35–40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility.Abbreviations Da dalton - M_r relative molecular mass - Pr, Pfr red and far-red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986     

Generation and analysis of mice lacking the chemokine fractalkine

Fractalkine (CX(3)CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX(3)CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX(3)CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses of fractalkine(-/-) mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

Unique role of the chemokine domain of fractalkine in cell capture. Kinetics of receptor dissociation correlate with cell adhesion

The chemokine fractalkine (FK) has two structural features that make it unique in the chemokine family: a CX(3)C motif and an extended carboxyl terminus that anchors it to the cell surface. This mucin-like stalk or an equivalent spacer is required for FK to mediate the adhesion of cells expressing its receptor, CX(3)CR1. To determine whether the ability of FK to act as a cell adhesion molecule is due to the unique presentation of a chemokine domain on a stalk or to properties of the chemokine domain itself, we created a series of chimeras in which other soluble chemokines (RANTES (regulated on activation normal T cell expressed), monocyte chemoattractant protein 1, macrophage inflammatory protein 1 beta, secondary lymphoid tissue chemokine, and interleukin 8) were fused to the mucin stalk. When tested in a static-cell adhesion assay, many of these chemokine chimeras demonstrated activity equivalent to that of FK. In flow assays, however, none of the chimeras captured cells as efficiently as FK. Interestingly, FK captured cells expressing either CX(3)CR1 or the viral receptor US28. Cells bound to FK without rolling or detaching, whereas the interleukin 8 and monocyte chemoattractant protein 1 chimeras induced primarily cell rolling and detaching, respectively. In binding studies, FK has a significantly slower off-rate from its receptors than any of the other chemokine chimeras had for their cognate receptors. We conclude that presentation of a chemokine atop a mucin-like stalk is not, in and of itself, sufficient to capture cells. The unique ability of FK to mediate adhesion under flow may be a function of its slow receptor off-rate.     

Phenotypic characteristics of human monocytes undergoing transendothelial migration

In our study we characterised the immunophenotype of monocytes that migrated through an endothelial cell (EC) monolayer in vitro. We found that monocyte migration led to an enhanced expression of CD11a, CD33, CD45RO, CD54 [intercellular cell-adhesion molecule (ICAM)-1] and human leucocyte antigen-DR. The most striking increase was observed for ICAM-1 when ECs were activated with tumour necrosis factor-α and interleukin-1α. The results of our study indicate the following: (1) there is a characteristic immunophenotype on the surface of monocytes after transendothelial migration; (2) this phenotype seems to be induced by interactions between monocytes and ECs; and (3) this change is enhanced by the pretreatment of ECs with cytokines. Taken together, the results suggest that local cytokine production activating ECs is sufficient to enhance monocyte migration and that this, in turn, can induce changes consistent with an activated phenotype known to be interactive between antigen-presenting cells and T cells. These results have implications for our pathogenetic insights into rheumatoid arthritis.     

Peptide fragments from the tuftsin containing domain of immunoglobulin G synthesis and biological activity

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [3H-Arg4]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cell's capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsin's receptors.     

The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia

Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and 3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS.     

Peptide repertoire of human cerebrospinal fluid: novel proteolytic fragments of neuroendocrine proteins

Polypeptides in human cerebrospinal fluid (CSF), isolated by phase separation in chloroform–methanol–water and reversed-phase HPLC, were characterised by sequence analysis and mass spectrometry. This identified the presence of peptide fragments of testican, neuroendocrine specific protein VGF, neuroendocrine protein 7B2, chromogranin B/secretogranin I, chromogranin A, osteopontin, IGF-II E-peptide and proenkephalin. The majority of these fragments were generated by proteolysis at dibasic sites, suggesting that they are derived by activities related to prohormone convertase(s). Several of the fragments have previously not been detected, and their functions in CSF or elsewhere are unknown. A characteristic feature of all these fragments is a very high content of acidic residues, in particular glutamic acid. In addition to the fragments of neuroendocrine proteins, endothelin-binding receptor-like protein 2, ribonuclease 1, IGF-binding protein 6, albumin, α₁-acid glycoprotein 1, prostaglandin-H2 -isomerase, apolipoprotein A1, transthyretin, β₂-microglobulin, ubiquitin, fibrinopeptide A, and C4A anaphylatoxin were found.     

Peptide affinity chromatography of human clotting factor VIII: Screening of the vWF-binding domain

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.     

Influence of surgery on in-vitro cytokine production by human monocytes

Surgery leads to significant modulation of the immune system, in which cytokines play a major role. Circulating interleukin 6 (IL-6) and IL-1 have been reported following surgery whereas tumor necrosis factor alpha (TNF-alpha) is only found in gut ischemia-associated surgery. We have investigated the consequences of surgery on in-vitro cytokine production by human monocytes stimulated by lipopolysaccharide (LPS) and staphylococcal toxic shock syndrome toxin-1 (TSST-1). Comparisons were made between the responsiveness of cells obtained the day before (D-1), during (D0) and after (D1, D2, D3) surgery. Patients undergoing abdominal aortic surgery (N = 9), carotid surgery (N = 4) and spinal surgery (N = 4) have been studied. A significant decrease of TNF-alpha, IL-1 beta and IL-1 alpha production by monocytes prepared from blood samples taken during the surgery was noticed, whereas IL-6 production was not significantly modified. On D2 a significant increase of monocyte responsiveness was observed and levels of cytokine productions rose back to initial values by the end of the follow up. The diminished in-vitro cytokine production observed during surgery might be the consequence of the effects of anaesthetic drugs, whereas the enhancement observed on D2 might reflect the surgical stress, leading to in-vivo priming of circulating monocytes.     

Absence of induction of IL-1 production in human monocytes by complement fragments

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.     

The effect of vinblastine, colchicine and hexylene glycol on migration of human monocytes

Human monocytes treated with the microtubule-disrupting agents vinblastine and colchicine show enhanced migration into micropore filters. When treated with hexylene glycol, which appears to stabilize microtubules, their migration is inhibited. These results suggest that disruption of the microtubules may facilitate deformation of the cell necessary for migration through small holes and that stabilization of the microtubules may increase cell rigidity and inhibit such migration.