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Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage-myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A 2B adenosine receptor (A 2B AR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A 2B AR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A 2B AR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Peptide amphiphile micelles (PAMs) are a nanoparticle platform that have gained popularity for their targeting versatility in a wide range of disease models. An important aspect of micelle design is considering the type of hydrophobic moiety used to synthesize the PAM, which can act as a contributing factor regarding their morphology and targeting capabilities. To delineate and compare the characteristics of spherical and cylindrical micelles, we incorporated the monocyte-targeting chemokine, monocyte chemoattractant protein-1 (MCP-1), into our micelles (MCP-1 PAMs). We report that both shapes of nanoparticles were biocompatible with monocytes and enhanced the secondary structure of the MCP-1 peptide, thereby improving the ability of the micelles to mimic the native MCP-1 protein structure. As a result, both shapes of MCP-1 PAMs effectively targeted monocytes in an in vitro binding assay with murine monocytes. Interestingly, cylindrical PAMs showed a greater ability to attract monocytes compared to spherical PAMs in a chemotaxis assay. However, the surface area, the multivalent display of peptides, and the zeta potential of PAMs may also influence their biomimetic properties. Herein, we introduce variations in the methods of PAM synthesis and discuss the differences in PAM characteristics that can impact the recruitment of monocytes, a process associated with disease and cancer progression.

Triple-negative (TN) breast cancers (ER - PR - HER2 - ) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance.

Inflammation is the normal immune response of vascularized tissues to damage and bacterial products, for which leukocyte transendothelial migration (TEM) is critical. The effects of cell-to-cell contact seen in both leukocyte and endothelial cells include cytoskeleton rearrangement, and dynamic expression of adhesion molecules and metalloproteinases. TEM induces expression of anti-apoptotic molecules, costimulatory molecules associated with antigen presentation, and pattern recognition receptors (PRR), such as TLR-4, in monocytes. However, little is known about how TLR-4 increment operates in monocytes during an inflammatory response. To understand it better, we used an in vitro model in which monocytes crossed a layer of IL-1β stimulated Human Umbilical Vein Endothelial Cells (HUVEC). After TEM, monocytes were tested for the secretion of inflammatory cytokines and chemokines, their phenotype (CD14, CD16, TLR-4 expression), and TLR-4 canonical [Nuclear Factor kappa B, (NF-κB) pathway] and non-canonical [p38, extracellular signal-regulated kinases (ERK) 1/2 pathway] signal transduction induced by lipopolysaccharide (LPS). Phagocytosis and bacterial clearance were also measured. There was diminished secretion of LPS-induced inflammatory cytokines (IL-1β, IL-6, and TNF-α) and higher secretion of chemokines (CXCL8/IL-8 and CCL2/MCP-1) in supernatant of TEM monocytes. These changes were accompanied by increases in TLR-4, CD14 (surfaces expression), p38, and ERK1/2 phosphorylated cytoplasmic forms, without affecting NF-κB activation. It also increased bacterial clearance after TEM by an O2 -independent mechanism. The data suggest that interaction between endothelial cells and monocytes fine-tunes the inflammatory response and promotes bacterial elimination., (© 2015 International Federation for Cell Biology.)

Aims: We aimed to characterize the influence of acute myocardial infarction (AMI) on the metabolic activity of the bone marrow (BM) and on the composition and functional activity of BM-derived mononuclear cells (BMC). Acute ischaemia or other stressors induce the mobilization of progenitor cells from the BM stem cell niche. The effect of AMI on the numbers and functional activity of cells within the BM is unknown., Methods and Results: In patients of the REPAIR-AMI trial as well as in mice, the number and functionality of BMC was compared with respect to the time interval from AMI. Activation of Wnt signalling was assessed after AMI induction in TOP-GAL transgenic reporter mice, carrying a β-galactosidase gene driven by an LEF/TCF/β-catenin responsive promoter. The metabolic activity of the BM, as determined by F-18-fluorodeoxyglucose-positron emission tomography, was significantly higher in patients with AMI compared with patients with chronic post-ischaemic heart failure. Moreover, the number of haematopoietic CD34(+) (P < 0.05) and CD133(+) (P < 0.05) cells in the BM aspirates was significantly increased in patients within 7 days after AMI. In order to confirm these clinical data, we induced AMI in mice, which time-dependently increased the number of c-kit + Sca-1 + lin- cells and colony-forming units in the BM. Activation of the BM by AMI induced a significant increase in Wnt signalling, which is known to induce proliferation of haematopoietic stem cells, and demonstrated increased levels of the Wnt target Axin-2 in BM-derived cells on Day 7 (P < 0.01 vs. control)., Conclusion: Acute myocardial infarction is associated with an increased metabolic activity and increased levels of progenitor cells within days after AMI. These findings document an activation of the stem cell niche within the BM following AMI, which may have important implications for the optimal timing of cell aspirations used for therapeutic application in patients with AMI.

Increased neutrophil chemotaxis and hyperresponsiveness to streptococci have been considered to play a role in Behçet's disease (BD) pathogenesis. Our aim was to correlate TLR2 expression and chemotactic responses stimulated by bacterial lipoteichoic acid (LTA) in BD neutrophils. Thus, we assessed expressions of TLR2 and the correlate receptors CD14, CD114 (G-CSF receptor), CD116 (GM-CSF receptor) and also TLR4 on circulating neutrophils and monocytes of patients with active BD. Serum concentration of soluble CD14 (sCD14) was also measured. Neutrophil chemotactic responses from BD patients and healthy controls under LTA stimulation were assessed. Disease activity was evaluated by Behçet's Disease Current Activity Form (BDCAF). Receptor expressions were measured by flow cytometry, neutrophil chemotaxis was assessed in a Boyden chamber and sCD14 was measured by enzyme-linked immunosorbent assay. TLR2 expression was higher only on BD monocytes compared with healthy controls (39.9 +/- 13.1 vs. 33.6 +/- 5.3, p = 0.019). Expressions of all other receptors were similar in BD and control group. Of particular interest, TLR2 expression on neutrophils was similar in both groups and, when stimulated with LTA, BD neutrophils showed chemotactic responses similar to the controls. Neutrophils exhibited increased chemotaxis only when incubated with BD plasma. Serum sCD14 concentration was higher in BD patients than in controls (1,920.8 +/- 563.6 vs. 1,623.2 +/- 391.3 ng/ml, p = 0.008), and it was positively correlated with both CD14 expression on circulating monocytes membrane (p = 0.035) and BDCAF scores (p = 0.025). In conclusion, isolated BD neutrophils do not overexpress TLR2 neither overreact to LTA. However, because TLR2 expression was higher on BD monocytes, sCD14 from monocyte origin correlated with disease activity and neutrophil hyperchemotaxis occurred on strict dependence of plasmatic soluble factors, we suggest a possible role for bacterial stimulation of monocytes via TLR2 producing neutrophil-stimulating pro-inflammatory factors in BD.

Monocytes/macrophages recruited into the arterial wall during atherogenesis are crucial in the initiation and progression of atherosclerosis and play a fundamental role in the destabilization process that is the main causal event of acute coronary syndromes. In the present study, we investigated the effect of the mammalian target of rapamycin inhibitor everolimus on macrophage accumulation within carotid lesions elicited by perivascular collar placement in cholesterol-fed rabbits. Everolimus (1.5 mg/kg given 1 day before collaring followed by 1 mg/kg/day for 14 days, administered by oral gavage) markedly decreased lesion macrophage content as compared with vehicle control (-65%; p < 0.01). This effect was associated with a reduction in intimal thickening and occurred in the absence of changes in plasma cholesterol concentrations. To gain insights on the potential mechanism(s) underlying this effect, we investigated the influence of everolimus on chemoattractant-induced migration of human monocytes in vitro. Pretreatment with therapeutic concentrations of everolimus (10 nM) significantly lowered monocyte chemotaxis in response to various chemotactic factors (i.e., monocyte chemoattractant protein-1/CCL2, fractalkine/CX3CL1, interleukin-8/CXCL8, complement fragment 5a, or N-formyl-Met-Leu-Phe) without inducing monocyte cell death. These results suggest that everolimus may favorably influence the atherosclerotic process by affecting the recruitment of monocytes into early lesions.

During inflammation, monocytes roll on activated endothelium and arrest after stimulation by proteoglycan-bound chemokines and other chemoattractants. We investigated signaling pathways downstream of G protein-coupled receptors (GPCRs) that are relevant to alpha4beta1 integrin affinity up-regulation using formyl peptide receptor-transfected U937 cells stimulated with fMLP or stromal-derived factor-1alpha and human peripheral blood monocytes stimulated with multiple chemokines or chemoattractants. The up-regulation of soluble LDV peptide or vascular cell adhesion molecule-1 (VCAM-1) binding by these stimuli was critically dependent on activation of phospholipase C (PLC), inositol 1,4,5-triphosphate receptors, increased intracellular calcium, influx of extracellular calcium, and calmodulin, suggesting that this signaling pathway is required for alpha4 integrins to assume a high-affinity conformation. In fact, a rise in intracellular calcium following treatment with thapsigargin or ionomycin was sufficient to induce binding of ligand. Blockade of p44/42 and p38 mitogen-activated protein (MAP) kinases, phosphoinositide 3-kinase, or protein kinase C (PKC) signaling did not inhibit chemoattractant-induced LDV or VCAM-1 binding. However, activation of PKC by phorbol ester up-regulated alpha4beta1 affinity with kinetics distinct from those of GPCR signaling. A critical role for PLC and calmodulin was also established for leukocyte arrest and adhesion strengthening.

We examined the behavior of polymorphonuclear leukocytes (PMNs) and monocytes during subcutaneous plasma extravasation in guinea-pigs. Plasma extravasation was induced by intradermal injection of zymosan-activated plasma (ZAP). The degree of extravasation correlated logarithmically with the concentration of injected ZAP, and was composed of PMN-dependent and -independent components. The latter was mediated primarily by histamine. The former accounted for 40-50% of the total plasma extravasation, peaked within 15 min, and then rectilinearly decreased with a half-life between 30 and 40 min. Histological examination of skin at 15 min after ZAP injection demonstrated PMN attachment to the luminal surface of venule endothelial cells, without evidence of PMN extravasation. We next examined whether monocyte infiltration of subcutaneous tissue played a causal role in plasma extravasation. Monocyte-predominant infiltration was initially caused by an intradermal injection of a monocyte-specific chemotactic factor, the S19 ribosomal protein (RP S19) dimer. Monocyte infiltration did not induce plasma extravasation even in guinea-pigs with elevated peripheral blood monocyte levels following administration of a macrophage-colony stimulating factor. A simultaneous injection of prostaglandin E2, a vasodilating agent, with RP S19 dimer also did not induce plasma extravasation. In contrast, a simultaneous injection of RP S19 dimer with ZAP changed the leukocyte infiltration pattern from PMN-predominant to monocyte-predominant, and almost completely suppressed the PMN-dependent component of the ZAP-induced plasma extravasation. The lack of plasma extravasation in the monocyte-predominant pattern was reproduced when a strong monocyte infiltration was induced by an intradermal injection of apoptotic cells. We conclude that leukocyte-induced plasma extravasation is specific for PMN, and is not due to a physical leakage of plasma during leukocyte emigration. Rather, plasma extravasation is probably caused by a cognate interaction between PMNs and postcapillary venule endothelial cells.

Objective: Leukotriene B4 (LTB4) has been implicated in the trafficking of monocytes to inflammatory pathologic conditions, such as transplant rejection and atherosclerosis. The aim of this study was to determine the mechanisms by which LTB4 contributes to monocyte capture from the circulation., Methods and Results: In in vitro and in vivo vascular models, the lipid chemoattractant LTB4 was an equipotent agonist of monocyte adhesion compared with the chemokine monocyte chemoattractant protein-1 (MCP-1). Adenoviral gene transfer of specific endothelial adhesion molecules and blocking monoclonal antibody studies demonstrated that LTB4 triggers both beta1- and beta2-integrin-dependent adhesion. Flow cytometry studies suggested that changes in integrin avidity or affinity, rather than alterations of integrin surface expression, were responsible for the chemoattractant-triggered arrest. Surprisingly, in contrast to the peptide chemokine MCP-1, LTB4 did not activate the phosphoinositide 3-kinase pathway, which is a functionally critical step in chemokine-triggered effector functions., Conclusions: LTB4 is a potent trigger of monocyte adhesion under flow yet mediates its effects via pathways that appear to differ from peptide chemoattractants. A better understanding of the mechanisms of LTB4-induced monocyte trafficking might shed insight into disease pathogenesis and pinpoint critical steps for therapeutic intervention for multiple human inflammatory pathologic conditions.

1. The present study was aimed to investigate the effect of benzydamine, an anti-inflammatory drug devoid of activity on arachidonic acid metabolism, on monocyte chemotaxis and to define the possible biochemical correlates of activity. 2. Benzydamine inhibited monocyte chemotaxis in response to three classes of chemoattractants: the prototypic CC-chemokine CCL2 (MCP-1), the microbial product fMLP and the complement cascade component C5a. The effect was dose-dependent with IC50's of 100, 50 and 45 microm for MCP-1/CCL2, fMLP and C5a, respectively. At the dose of 100 microm, the effect resulted in a 50+/-10% inhibition of MCP-1/CCL2-induced chemotaxis and 53+/-6 and 54+/-5% inhibitions of chemotaxis in response of fMLP and C5a, respectively (n=3). 3. Receptor expression as well as calcium fluxes in response to chemoattractants were not affected by benzydamine. 4. Benzydamine strongly inhibited chemoattractant-induced activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and of its upstream activator kinase MEK1/2. ERK1/12 activation in response to chemoattractants was 89-98% inhibited by a 100 microm concentration of benzydamine with an IC50 of 30 microm. 5. Under the same experimental conditions, pretreatment with 100 microm benzydamine caused a 75-89% inhibition of p38 activation (IC50 25 microm). 6. These results indicate that the anti-inflammatory activity of benzydamine is exerted at multiple levels, including monocyte migration to chemotactic factors associated to a blockage of ERK and p38 MAPK pathways.

Bone resorption is regulated by the immune system, where receptor activator of nuclear factor (NF)kappaB ligand (RANKL), a new member of the tumor-necrosis factor family, may contribute to pathological conditions. Due to the role of RANKL in the maturation of monocyte-derived osteoclasts, we hypothesized that RANKL could exert chemotactic properties toward monocytic cells. Our results demonstrate that RANKL induces the migration of MonoMac-6 monocytic cells as well as human freshly isolated total peripheral blood mononuclear cells (PBMC) and CD14+ purified PBMC. RANKL induces the migration of MonoMac-6 cells in a dose-dependent manner and with an efficacy similar to MCP-1. After an 8-h incubation, the soluble form of RANKL (sRANKL) started to exhibit a chemoattractive effect on MonoMac-6 cells, with an increased effect observed up to 24 h. RANKL elicits an additive chemotactic effect to MCP-1. Furthermore, addition of the RANKL decoy receptor osteoprotegerin in the lower well or RANKL in the upper well abrogates the RANKL-induced migration of MonoMac-6 cells, hallmarking a true specific activity. RNase protection assay experiments indicate that exposure of MonoMac-6 cells to RANKL had no significant effect on the expression of a variety of chemokines, known to attract monocytes. This study provides evidence that RANKL behaves as a chemotactic factor for monocytic cells, emphazing the cross-talk between bone and immune systems.

Endothelins (ET-1, ET-2 and ET-3) are 21-amino acid vasoactive peptides that bind to G-protein-linked transmembrane receptors, ET-RA and ET-RB. As well as modulating vasoconstriction, endothelins regulate growth in several cell types and may also affect differentiation, inflammation and angiogenesis. Both macrophages and endothelins are found in areas of hypoxia in solid tumors and ET-2 expression may be modulated by hypoxia in some tumors. As the peptide structure of mature endothelins is similar to that of CXC chemokines, we asked if endothelins contribute to control of macrophage distribution in tumors. We found that ET-2 is a chemoattractant for macrophages and THP-1 monocytic cells, but not for freshly isolated monocytes. The chemotactic response to ET-2 shows a typical bell-shaped response curve. Experiments with endothelin receptor antagonists showed that migration to ET-2 is mediated via the ET-RB receptor. Moreover, monocytes do not express ET-RB. Chemotaxis towards ET-2 is via the MAPK pathway: p44 and p42 are phosphorylated when THP-1 cells are stimulated with ET-2, and the MAPKK inhibitor PD98059 stops chemotaxis. As with 'classical' chemokines, migration toET-2 is also inhibited by hypoxia and by pertussis toxin. As well as its chemotactic properties, ET-2 leads to activation of macrophages. In human breast tumors that express ET-2, endothelins and ET-RB expressing macrophages often co-localized. While shorter than 'classical' chemokines, ET-2 shares a similar peptide sequence with chemokines and may signal via a similar receptor and MAPK-mediated pathway. Furthermore, ET-2 expression by tumors may modulate the behavior of macrophages such that activated cells accumulate in areas of hypoxia.

Background: Histamine is a well-known mediator eliciting different responses in immune and nonimmune cells, but its role in modulating dendritic cell (DC) functions has been marginally investigated., Objective: The purpose of this investigation was to analyze whether human monocyte-derived DCs express functional histamine receptors according to their maturation stage., Methods: DCs were derived from monocytes and used as immature or LPS-differentiated cells. DCs were tested for histamine receptor expression, chemotaxis, cytokine release, and the capacity to induce T-cell differentiation in response to specific histamine receptor agonists., Results: Immature and mature DCs expressed the mRNA for H1, H2, and H3 histamine receptors. Histamine induced intracellular Ca2+ transients, actin polymerization, and chemotaxis in immature DCs. Maturation of DCs resulted in the loss of these responses. In maturing DCs, however, histamine dose-dependently enhanced intracellular cAMP levels and stimulated IL-10 secretion while inhibiting production of IL-12. As a consequence, histamine might contribute to the impairment of generation of allogeneic type 1 responses via maturing DCs. Specific histamine receptor agonists or antagonists revealed that Ca2+ transients, actin polymerization, and chemotaxis of immature DCs were due to stimulation of H1 and H3 subtypes. Modulation of IL-12 and IL-10 secretion by histamine involved the H2 and H3 receptors exclusively., Conclusions: Our study suggests that histamine has important biological effects on DC activities, opening the possibility that histamine released during inflammatory or immune responses could regulate DC functions and ultimately favor type 2 lymphocyte-dominated immunity.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.

Blood monocytes of patients with thyroid autoimmune disease (TAID) display defects in rearranging their cortical actomyosin cytoskeleton ('polarize') in response to chemoattractants. Such rearrangements also take place after the adherence of monocytes to the extracellular matrix (ECM). It is therefore not surprising that monocytes are primed after fibronectin (FN) adherence, displaying an enhanced polarization toward chemoattractants. We investigated the integrin expression and chemoattractant-induced polarization of monocytes of TAID patients before and after FN adherence. Since cytoskeletal rearrangements are also required during the transition of monocytes into veiled antigen-presenting cells (VCs), we investigated such transition of FN-adherent monocytes of TAID patients. Adherent and nonadherent monocyte populations from TAID patients and healthy controls were subjected to a polarization test with the chemoattractant fMLP (or MCP-1), FACS analyses (FITC-labelled FN, CD29, CD49e, d, b and a) and tested for their capability to develop into veiled APC. Monocytes of healthy individuals showed an improved chemoattractant-induced cell polarization after FN adherence, not reflected by TAID monocytes, in which chemoattractant-induced polarization worsened. Monocytes of healthy individuals up-regulated CD49e and d integrins and their capability to bind FITC-labelled FN after adherence to a FN-coated plate, as well as enhancing their capability to generate T cell-stimulatory VCs. Monocytes of TAID patients did not. These data indicate that integrin- (and chemokine-) mediated functions are hampered in monocytes of TAID patients. Because integrin action is pivotal to processes such as monocyte adherence to endothelial cells, uropod formation, migration into tissues and differentiation into APC and macrophages, these defects might underly immune dysbalances important in thyroid autoimmune development.

Chemoattractants and chemokines induce arrest of rolling monocytes during emigration from blood into tissues. In this study, we demonstrated that alpha4 integrin affinity for vascular cell adhesion molecule (VCAM)-1 was upregulated rapidly and transiently by chemoattractants and stromal cell-derived factor (SDF)-1alpha and mediated monocyte arrest. alpha4 integrin affinity changes were detected and blocked using soluble VCAM-1/Fc (sVCAM-1/Fc). In a flow cytometry assay, markedly increased sVCAM-1/Fc binding to human blood monocytes or U937 cells transfected with formyl peptide (FP) receptor was detected 30 s after FP or SDF-1alpha treatment and declined after 2 min. In a parallel plate flow chamber assay, FP, C5a, platelet-activating factor, or SDF-1alpha coimmobilized with VCAM-1 induced leukocyte arrest, which was blocked by inclusion of sVCAM-1/Fc but not soluble nonimmune immunoglobulin G in the assay buffer.

The intracellular signaling pathways involved in human monocyte chemotaxis toward a variety of chemoattractant molecules were evaluated using selected pharmacological agents. Neither phosphatidylinositol-3-kinase (P13K) or extracellular signal-regulated kinase (ERK) activity were required for monocyte migration toward monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), macrophage inflammatory protein-1alpha (MIP-1alpha) or formyl-Met-Leu-Phe (fMLP), since pretreatment with wortmannin or LY294002, or with PD098059, had no effect on the chemotactic response. Addition of forskolin and IBMX significantly attenuated chemotaxis to each of these chemoattractants and was reversed by co-treatment with Rp-cAMP, a competitive inhibitor of cAMP-dependent protein kinase A. Incubation with the protein kinase C (PKC) inhibitor GF109203X-HCl (GF109) did not affect monocyte migration, but pretreatment of monocytes with PMA significantly impaired the response to each of these chemotactic agents. Inhibition by PMA was reversed by co-treatment with GF109, implying that heterologous PKC activation is capable of desensitizing chemokine and fMLP-induced monocyte chemotaxis. These results help to define the signalling pathways involved in human monocyte chemotaxis and suggest pharmacological approaches to evaluating the cross-desensitization of chemoattractant-induced leukocyte migration.

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-alpha (MGSA/GROalpha), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROalpha was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.

Background: We established a T cell line, STO-5, which constitutively produced monocyte/macrophage chemotactic activity via human T cell lymphoma-leukemia-virus-induced transformation of normal human T cells., Methods: We isolated and purified a lactose-binding protein, MCF-pl5-L (MW of about 50 kD, pl of about 5) from a conditioned medium of STO-5. By using highly purified MCF-pl5-L, its biological and physicochemical properties were elucidated in comparison with C5a and MCP-1., Results: MCF-pl5-L exhibited an evident dose-dependent monocyte chemotactic activity (MCA). MCF-pl5-L had no or little affinity for heparin unlike chemokines such as MCP-1. We further found that MCF-pl5-L exhibited potent chemotactic activity not only for monocytes but also for alveolar macrophages. In contrast, C5a and MCP-1 failed to show evident chemotactic activity for alveolar macrophages though they did show MCA. MCF-pl5-L failed to exhibit evident eosinophil and neutrophil chemotactic activities, indicating its chemotactic activity is selective for monocytes/macrophages. Regarding the biological functions of MCF-pl5-L other than MCA and chemotactic activity for alveolar macrophages, we found that MCF-pl5-L but not C5a and MCP-1 could prolong the life span of alveolar macrophages, probably by inhibiting apoptosis of macrophages, and stimulate the production of TNF-alpha from macrophages., Conclusions: These results suggest that MCF-pl5-L plays a role as an immune modulator for monocytes/macrophages in the site., (Copyright 2000 S. Karger AG, Basel)

Chymase is a major chymotrypsin-like serine protease expressed in the secretory granules of mast cells in many mammalian species. In this study, we revealed the chemotactic activity of chymase for human mononuclear cells and neutrophils with a 48-well microchemotaxis chamber technique. Human chymase showed the potent chemotactic activity for monocytes and neutrophils dose-dependently in a concentration range from 0.1 to 10 microg/mL, corresponding to about 4-400 microM. The activity was as potent as that of N-formyl-methionyl-leucyl-phenylalanine. Chymase also stimulated cell migration of lymphocytes and purified T cells, but checkerboard analysis revealed that the effect was chemokinetic rather than chemotactic. Inhibition of chymase activities with chymase inhibitors, such as antileukoprotease and Bowman-Birk soybean trypsin inhibitor, significantly inhibited the chemotactic activity of chymase, suggesting that the proteolytic activity of chymase participates in the chemotactic activity. Our results suggest that mast cell chymase acts as a chemoattractant, and may play a role in the accumulation of inflammatory cells in development of the chronic inflammatory responses of allergic and nonallergic diseases.

Macrophages in lymphoid organs are in close contact to nerve terminals of the sympathetic nervous system. Hence, these cells could be targets of neuronal modulation. We studied sympathetic neurotransmitters as chemoattractants enabling the aggregation of macrophages and nerve terminals. Norepinephrine (NE), neuropeptide Y (NPY), isoproterenol (beta-adrenergic), p-aminoclonidine (alpha2-adrenergic), methoxamine (alpha1-adrenergic), and adenosine triphosphate (ATP) were used to study human monocyte and macrophage migration in 48-well Boyden chambers. NE stimulated chemotaxis of monocytes and macrophages at an optimal concentration of 10-(10) M (P < 0.025). Isoproterenol, but not p-aminoclonidine or methoxamine, induced chemotaxis of monocytes (10(-10) M, P < 0.05). In these studies, elevation of cAMP is a critical step in NE-induced chemotaxis of monocytes. NPY (10(-11) M, P < 0.05) stimulated monocyte chemotaxis as well. ATP at 10(-4) and 10(-5) M stimulated undirected cell mobility (P < 0.05). All tested neurotransmitters of the sympathetic nerve terminal were potent chemoattractants. These findings may explain the close association of nerves and macrophages in tissue and lymphoid organs and may thus be of functional relevance in neuroimmunomodulation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances and primes monocyte functions, but its role in monocyte migration is poorly understood. We examined monocyte migration across human umbilical vein endothelial cells (HUVEC) grown on filters. GM-CSF had no chemotactic or chemokinetic effect. However, GM-CSF enhanced monocyte transendothelial migration (TEM) through unstimulated and IL-1-activated (5 h) HUVEC in response to C5a or monocyte chemoattractant protein-1 in a dose-dependent fashion, increasing the migration from 28.7 +/- 5.3% to 41.8 +/- 6.2% (n = 8, p < 0.05) and from 34.8 +/- 6% to 50.3 +/- 3.1%, p < 0.05), respectively. The enhanced TEM was inhibited by monoclonal antibodies (mAb) to LFA-1, but not by mAb to Mac-1 or to VLA-4. Furthermore, GM-CSF up-regulated and activated LFA-1, as assessed by NKI-L16 neoepitope expression. The results indicate that: (1) GM-CSF can prime monocytes for increased TEM, (2) GM-CSF enhances LFA-1-mediated monocyte TEM and (3) this effect is in part mediated by increasing LFA-1 expression and activation. Thus, increased GM-CSF production may promote monocyte accumulation in inflammation not only by inducing monocytosis, but also enhancing migration.

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.

Within blood vessels the accumulation of monocytes/macrophages at sites of modified lipoproteins is an important feature in atherosclerosis. Recently the presence of LDL and other proteins modified by hypochlorous acid (HOCl-LDL) was demonstrated in human atherosclerotic vessels and human inflammatory kidney disease by immunohistology and protein chemistry. Chemokines contribute to a specific and directed migration of inflammatory cells. IL-8 (alpha-chemokine) attracts mainly neutrophils and distinct T-cell subsets while MCP-1 (beta-chemokine) preferentially acts on monocytes/macrophages. In the present study it was postulated that HOCl-LDL may induce and amplify inflammatory reactions by the induction of chemokine synthesis in local monocytes. After exposure of human monocytes to HOCl-LDL, it was found that mRNA and protein of the chemokine IL-8 was strongly induced, while the chemokine MCP-1 was not. HOCl-LDL itself led to a chemotactic migration of neutrophils. A chemotactic response of human monocytes toward HOCl-LDL was not detectable. We propose that HOCl-LDL may represent a form of LDL modification in the atherosclerotic process which initiates leukocyte infiltration; these mononuclear cells have been observed in the early stages of atherosclerosis.

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5-1 microg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G-induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein-coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 microg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.

The beta-chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1alpha), MIP-1beta and regulated on activation, normal T cells, expressed and secreted (RANTES) induced the in vitro migration of the monocytic cell line MonoMac-6. MCP-1 exhibits the most potent chemotactic effect on this cell line while MIP-1alpha, RANTES and to a lesser extent MIP-1beta were more moderate inducers of cell migration. MonoMac-6 migration in response to chemokines was shown to be a chemotactic and not a chemokinetic response, which was inhibited by pertussis and cholera toxins suggesting a role for G proteins in chemokine receptor-mediated signalling in these cells; chemotaxis of MonoMac-6 cells in response to MCP-1 was abrogated by the addition of anti-MCP-1 antibody. The response of MonoMac-6 cells to the alpha-chemokines IL-8, IP-10, growth-related peptide (Gro) alpha and MIP-2beta was substantially weaker than to the beta-chemokines. MCP-1 caused an alteration in cellular morphology by increasing ruffling at the cell membrane and the number of cells exhibiting extended pseudopodia. The chemotactic response of MonoMac-6 cells to beta-chemokines was compared with less well-differentiated myelomonocytic cell lines. THP-1 showed a similar, but weaker response to the beta-chemokines while both HL60 and U937 failed to respond to any member of this subfamily when tested under the same conditions. These results suggest that the differentiation status of cells of monocytic lineage may affect their response to beta-chemokines.

We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a cGMP-dependent mechanism. Therefore, plasmin represents a proinflammatory activator for human monocytes.

The newly described products of 5-hydroxyeicosanoid dehydrogenase, 5-oxo-6,8,11,14-eicosatetraenoic acid (ETE) and 5-oxo-15(OH)ETE, induced directional migration and actin polymerization of human monocytes in vitro. At peak concentrations, the two eicosanoids had a chemotactic activity of about 40% of that observed in the presence of an optimal concentration of FMLP and twice the activity elicited by the related eicosanoid 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). 15-Oxo-ETE showed a very low but detectable chemotactic activity. All of these chemotactic responses were blocked by Bordetella pertussis toxin, but were resistant to LY255283, a leukotriene B4 (LTB4) receptor antagonist. 5-Oxo-ETEs and 5-HETE induced homologous desensitization of chemotactic response, but did not cross-desensitize to other chemotactic agonists (e.g., monocyte chemotactic protein (MCP)-1 and LTB4). 5-Oxo-ETEs increased in a synergistic fashion the monocyte migration to MCP-1 and MCP-3. In the same range of concentrations, 5-oxo-ETE increased MCP-1-induced release of arachidonic acid from labeled monocytes. No synergistic interaction was observed when FMLP was used as chemoattractant. Thus, this study identifies monocytes as cells responsive to 5-oxo-ETEs and shows that monocyte activation by 5-oxo-ETEs occurs through an LTB4 receptor-independent mechanism that associates with pertussis toxin-sensitive G proteins. The synergistic interaction between 5-oxo-ETEs and C-C chemokines, two families of mediators both synthesized by phagocytic cells, may be relevant in vivo for the regulation of monocyte accumulation at sites of allergic and inflammatory reactions.

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall. Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients. The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments. Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.

The increase in intracellular free Ca2+ ([Ca2+]i) associated with interaction of monocyte chemotactic protein-1 (MCP-1) and related chemokines beta with adherent human blood monocytes was investigated at the single-cell level. We used f-MLP as reference chemotactic agent. MCP-1 caused an increase in [Ca2+]i in individual adherent monocytes, with 95% of cells responding to the chemokine at 20 ng/mL. Response to MCP-1 was already detectable at 1 pg/mL, whereas at least 5 ng/mL were required for significant chemotactic response. The kinetics of the increase in [Ca2+]i were considerably different for MCP-1 compared with f-MLP. MCP-1 produced a slow increase of [Ca2+]i that reached a plateau in 5 to 7 minutes. On the other hand, the increase of [Ca2+]i induced by f-MLP appeared to be biphasic, with a fast phase peaking after 5 to 40 seconds followed by a slower wave. Blocking of Ca2+ channels by Ni2+ or Cd2+ and/or chelation of extracellular free Ca2+ considerably reduced but did not abolish response to MCP-1, had no effect on the first wave of [Ca2+]i induced by f-MLP, and completely abrogated the second, slower wave. Thapsigargin, which empties intracellular Ca2+ stores, inhibited f-MLP-induced [Ca2+]i increase but fully blocked the action of MCP-1 only when combined with Ni2+. Thus, increase of [Ca2+]i induced by MCP-1 is apparently due to independent opening of a channel and mobilization from intracellular stores, whereas f-MLP-induced mobilization of Ca2+ from stores causes subsequent opening of a channel. At variance with MCP-1, the related chemokine MCP-2 induced only a low increase of [Ca2+]i in about 40% of adherent monocytes. Inhibition of chemokine-induced increase of [Ca2+]i by cholera or pertussis toxin indicated that MCP-1 and MCP-2 activate monocytes through different intracellular pathways. These results demonstrate at the single-cell level that the mechanisms and dynamics of increased [Ca2+]i are considerably different for f-MLP and chemokines beta. In addition, the [Ca2+]i increase induced by the two related chemokines beta MCP-1 and MCP-2 appears to be differently regulated, suggesting interaction with distinct receptors.

Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl-phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins.

Human monocytic THP-1 cells were differentiated to macrophages by incubation with 1.0 microM phorbol myristate acetate (PMA) for 1 to 18 h; cells were then assayed for the ability to migrate to MCP-1. In comparison to undifferentiated monocytes, the chemotactic response of PMA-differentiated cells to MCP-1 decreased with treatment time. This loss of the chemotactic response to MCP-1 correlated with increased in cellular enzymes characteristic of differentiated macrophages. Receptors binding assays demonstrated a parallel decrease in specific binding of MCP-1 with increased incubation with PMA. Undifferentiated monocytes had 1175 +/- 387 receptors per cell with a Kd of 1.53 +/- 0.35 nM. Cells differentiated to macrophages with PMA rapidly lost the ability to bind MCP-1, with a significant decrease apparent following 3 h incubation with PMA. The reduction in specific binding of MCP-1 by M phi-THP-1 cells was due to a decrease in both receptor number and affinity; receptor number was reduced to 481 +/- 106 receptors/cells with a Kd of 3.16 +/- 0.7 nM on cells treated for 3 h with PMA. The demonstrated changes in receptor affinity and expression with differentiation may be a mechanism of controlling macrophage responsiveness to chemokines in inflammatory foci.

Monocytes and polymorphonuclear leukocytes (PMNLs) migrate across cytokine (interleukin-1, tumor necrosis factor) activated endothelium or unstimulated endothelium in response to chemotactic factors in vitro and in vivo utilizing the CD11/CD18 (i.e., beta 2 integrin) adhesion molecule complex. However, in vivo studies have suggested that under some conditions and/or in certain tissues, leukocyte migration can also proceed via CD11/CD18-independent mechanisms. Here we compared adhesion mechanisms involved in the migration of 51Cr-labeled blood monocytes and PMNLs across human umbilical vein endothelium (HUVE) monolayers. We observed that monocyte transendothelial migration was not inhibited by monoclonal antibody (mAb) to CD18, when the HUVE was activated with IL-1 and the chemotactic factor C5a induced the migration. This CD18-independent monocyte migration was blocked by treatment of the monocyte with mAb to beta 1 or alpha 4 integrin, suggesting that very late activation antigen 4 (VLA-4) on the monocyte served as the alternative migration mechanism. In contrast to monocytes, mAb to CD18 inhibited PMNL migration to C5a across IL-1-activated HUVE, but only by 66%, significantly less than with C5a alone (84%) or IL-1-activated HUVE alone (95%). The migration of anti-CD18 mAb-treated PMNLs was not inhibited by function-blocking mAbs to sialyl Lewisx, L-selectin, beta 1 or alpha 4 integrin, the beta 3-related leukocyte response integrin, IL-8, or platelet-activating factor (PAF) antagonists, alone or in combination. Antibody-blocking studies of the ligands on HUVE indicated that E-selectin may be partially involved in this CD18-independent PMNL migration but that ICAM-1, VCAM-1, PECAM-1, and P-selectin are not involved. Of several chemotactic factors tested, C5a and C5adesArg in activated plasma were the most active in inducing CD18-independent migration of PMNLs across IL-1-activated HUVE. These results demonstrate that (1) monocytes can utilize VLA-4 for optimal transendothelial migration and (2) PMNLs may have a novel CD18-independent migration mechanism that is activated by C5a in conjunction with one or more ligands on cytokine-activated endothelium. This may involve, in part, E-selectin interacting with a yet to be identified counterreceptor on PMNLs.

The effect of recombinant human IL-4, IL-10, and IL-13 on the chemotaxis and antitumor activity of human blood monocytes induced by monocyte chemotactic and activating factor (MCAF) was examined. MCAF alone did not induce monocyte-mediated cytotoxicity against human melanoma (A375-M) cells whereas it significantly enhanced the cytotoxicity by norMDP-stimulated monocytes. MCAF, unlike IFN-gamma, had no priming effect on monocyte activation by norMDP. MCAF acted with norMDP or LPS to enhance the production of both IL-1 beta and TNF-alpha. Enhanced cytotoxicity of monocytes stimulated with MCAF plus norMDP was reduced by IL-1 receptor antagonist and anti-TNF-alpha antibody. IL-4, IL-10, and IL-13 suppressed the generation of antitumor activity and cytokine production (IL-1 beta and TNF-alpha) of monocytes stimulated with MCAF plus norMDP or LPS. Chemotaxis of monocytes induced by MCAF was not affected by norMDP or any of the anti-inflammatory cytokines (IL-4, IL-10, and IL-13). Moreover, the pretreatment of monocytes with anti-inflammatory cytokines did not suppress monocyte-chemotaxis. These findings suggest that in vivo recruitment and anti-tumor expression of blood monocytes induced by MCAF may be differently regulated by anti-inflammatory cytokines in vivo.

Only a minor proportion of monocytes responds to chemoattractants. To test the possibility that chemoattractant-responsive monocytes have distinctive functional characteristics, we enriched or depleted monocyte preparations for cells having a proinflammatory (P) phenotype and tested their responses to biologically relevant chemoattractants. We prepared monocyte subpopulations by one of three independent techniques to minimize the chances of artifacts: 1) depletion of P monocytes by adherence to fibronectin; 2) enrichment for P monocytes by negative selection for HLA-DR antigen; and 3) flow cytometric sorting. We measured responsiveness of monocyte subpopulations to N-formyl-Met-Leu-Phe, C5a, zymosan-activated serum, and monocyte chemoattractant protein-1 by three parameters: 1) polarization, 2) actin polymerization, and 3) directed migration. With each chemoattractant and each parameter, there was a striking direct relationship between the responsiveness of the monocyte preparations and their content of P monocytes. Our data indicate that the capacity of monocytes to be recruited rapidly from the vasculature into sites of inflammation is a property of a subpopulation of monocytes with a distinctive, neutrophil-like proinflammatory phenotype.

Migration of monocytes to sites of inflammation involves a series of attachments and detachments to extracellular matrix proteins. We examined the capacity of a chemokine, monocyte chemoattractant protein-1 (MCP-1), to regulate attachment of human monocytes to laminin, collagen I, collagen IV, or fibronectin. MCP-1 increased monocyte attachment to laminin in a dose- and time-dependent manner and stimulated a lesser increase to the other matrix proteins. Function-blocking monoclonal antibodies (MAbs) to the integrin beta 2-subunit (CD18), including Fab' fragments and alpha M (CD11b) blocked > 70% of attachment, whereas MAbs to the beta 1-integrin subunit reduced attachment by < 30%. This suggests that the CD11b/CD18 integrin is the predominant molecule involved in adhesion of MCP-1-stimulated monocytes to laminin. The association of CD11b with F-actin illustrated by confocal microscopy further supports this concept. In contrast, when monocytes were stimulated with the beta 1-stimulatory MAb TS2/16, monocyte adhesion to laminin occurred through beta 1-integrins. Thus MCP-1 can stimulate monocyte attachment to laminin, and this process is mediated through beta 2-integrins, principally CD11b/CD18.

YGVG and GLVPG, two new chemokinetic peptides, were identified in elastolytic digests of elastin, besides the known chemoattractant hexapeptide VGVAPG. In order to identify possible sequences responsible for the chemotactic and chemokinetic activities and to obtain structure-activity relationships we synthesized some analogues of these peptides: FGVG (an analogue of YGVG), GVAPG and VGAPG (derived from the hexapeptide by deletion of Val1 or Val3). FGVG has a higher chemotactic activity than YGVG (chemotactic indices of 0.62 and 0.49, respectively, at 10(-11) M) and is both chemotactic and chemokinetic. Checkerboard analysis demonstrated that both peptides derived from the hexapeptide present, in addition to the chemotactic activity, a chemokinetic activity. The chemotactic index of GVAPG is 0.66 at 10(-10) M, while for VGAPG it is 0.86 at 10(-9) M. These results indicate that the deletion of the N-terminal residue of the elastin chemotactic peptides, VGVAPG and GFGVG, gives rise to chemokinetic activity. CD and NMR studies showed that all peptides are largely unordered in aqueous solution.

Serum amyloid A (SAA) is an acute phase protein that in the blood is bound to high density lipoproteins; SAA is secreted mainly by hepatocytes, and its concentration increases in the blood up to 1000 times during an inflammatory response. At present, its biological function is unclear. Since some forms of secondary amyloidosis are caused by deposition in tissues of peptides derived from the SAA and leukocytes seem to be involved in this process, we investigated the effect of human SAA on human monocytes and polymorphonuclear cells (PMN). When recombinant human SAA (rSAA) was used at concentrations corresponding to those found during the acute phase (> 0.8 microM), it induced directional migration of monocytes and polymorphonuclear leukocytes. Preincubation of rSAA with high density lipoproteins blocked this chemoattractant activity for both monocytes and PMN. rSAA also regulated the expression of the adhesion proteins CD11b and leukocyte cell adhesion molecule 1 and induced the adhesion of PMN and monocytes to umbilical cord vein endothelial cell monolayers. When subcutaneously injected into mice, rSAA recruited PMN and monocytes at the injection site. On the basis of these data, we suggest that SAA may participate in enhancing the migration of monocytes and PMN to inflamed tissues during an acute phase response.

Monocyte chemoattractant protein-1 (MCP-1) is a monocyte-specific chemoattractant and activator and is a member of the chemokine-beta family of cytokines. To identify regions of MCP-1 which are required for its biological activity, we constructed human MCP-1 mutants that were expressed in eukaryotic cells and tested for their ability to attract monocytes in vitro. Deletion of amino acids 2-8 destroyed activity, suggesting that the amino-terminal region is necessary for activity. Within the deleted region, mutation of aspartate 3 to alanine produced a protein with 9% of wild-type activity, whereas mutation of asparagine 6 to alanine produced a protein with 52.9% of wild-type activity. Mutation of amino acids within the first intercysteine loop yielded variable results. Changing tyrosine 28 to aspartate or arginine 30 to leucine each produced proteins with essentially no monocyte chemoattractant activity. The side chains of these amino acids are predicted to point into a putative receptor binding cleft, and these loss-of-function mutations are consistent with this model. Also consistent is the retention of 60% of wild-type activity after mutation of serine 27 to glutamine, since the side chain of serine 27 is predicted to point away from the binding cleft. However, mutation of arginine 24, which lies outside of this area, to phenylalanine produced a protein with only 5% of wild-type activity, suggesting more complex interactions. Truncations of the carboxyl terminus, as well as mutation of aspartate 68 to leucine, generated proteins with 10-20% of wild-type activity. (Another carboxyl-terminal insertional mutation demonstrated that O-linked carbohydrate in MCP-1 alpha may be added to a threonine in the carboxyl-terminal region.) These findings are consistent with a structural model of dimeric MCP-1 which is similar to interleukin-8, in which amino acids that point into a cleft between the two carboxyl-terminal alpha-helices of the subunits are important for receptor binding. In addition, however, amino acids at the amino terminus and others outside of the interhelical cleft are also essential for activity. The carboxyl-terminal alpha-helix is not required for signaling per se but is required for maximal specific activity. Finally, four mutant proteins partially inhibited the ability of wild-type MCP-1 to attract monocytes in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

The biological responses of human monocytes and cells of the monomyelocytic THP-1 cell line to stimulation with members of the beta chemokine family are described in this report. All three chemokines tested, MCP-1, MIP-1 alpha, and RANTES, elicited mobilization of intracellular free calcium in monocytes and THP-1 cells. The magnitude of response was highest with MCP-1 stimulation. MCP-1 desensitized monocyte responses to MIP-1 alpha and RANTES, but no such desensitization was observed in THP-1 cells. MIP-1 alpha or RANTES did not desensitize either monocytes or THP-1 cells to MCP-1 stimulation. All three chemokines elicited a potent chemotactic response in monocytes that was comparable in magnitude to that of f-Met-Leu-Phe. MIP-1 alpha and RANTES required a fivefold higher dose than MCP-1 to elicit a peak response. On the contrary, THP-1 cells showed no significant chemotactic response. Studies of the desensitization of the monocyte chemotactic response indicated that all three chemokines are capable of causing complete homologous desensitization. Heterologous desensitization was observed only when monocytes were treated with MCP-1 followed by MIP-1 alpha or RANTES. Studies of actin polymerization and cell polarization responses of monocytes indicated that these two responses attained peak magnitude after 10 min of stimulation with any of the chemokines. Dose-response kinetics were similar to those of the chemotactic response. THP-1 cells again failed to show either of these two responses. Finally, the activation potential of the chemokines was measured by their ability to induce respiratory burst. A tenfold higher concentration than that causing peak chemotactic response was required to elicit respiratory burst and no heterologous desensitization was noticed. Respiratory burst could be induced in THP-1 cells with a direct protein kinase C activator but not with any of the chemokines. These results indicate that, of the three examples tested, MCP-1 is the most potent member of the beta chemokine family in the biological responses examined. Although a calcium response was elicited in THP-1 cells with chemokines, a lack of subsequent responses indicates some missing links in the downstream signal transduction pathways.

Gc-globulin has been found in bronchoalveolar lavage fluid in patients with chronic obstructive pulmonary disease (COPD) and adult respiratory distress syndrome (ARDS) and has been shown to enhance neutrophil chemotaxis to C5-derived peptides in vitro. We proposed that Gc-globulin may enhance the inflammatory response in lungs by influencing monocyte chemotaxis to C5-derived peptides as it does with neutrophils. Monocyte chemotaxis was measured in blind well chambers by a leading-front technique. Purified human Gc-globulin had no intrinsic chemotactic activity for monocytes at concentrations ranging from 1 fM to 1 microM. However, Gc-globulin, at concentrations as low as 10 pM, increased monocyte chemotaxis over 10-fold in a concentration-dependent fashion when added to non-chemotactic doses of C5a (0.1 nM) and C5a des Arg (0.5 nM). The chemotaxis-enhancing effect of Gc-globulin was specific for C5-derived peptides, as Gc-globulin did not enhance monocyte chemotaxis to other chemoattractants such as leukotriene B4 or formyl-Met-Leu-Phe. The enhancement of monocyte chemotaxis to C5-derived peptides by Gc-globulin was not a nonspecific effect of anionic proteins, as other serum proteins of similar size and charge did not enhance monocyte chemotaxis to C5a des Arg. These results indicate that Gc-globulin enhances the monocyte response to C5-derived peptides and, together with previous work, indicates that its presence in the airways of patients with COPD and ARDS may up-regulate the monocyte inflammatory response in the lungs.

Monocyte Chemotactic Protein-1 (MCP-1), a member of the Cys-Cys branch of the chemokine superfamily, induced a mepacrine- and manoalide-sensitive increase in the release of [3H]arachidonic acid from prelabeled human monocytes and monocytic THP-1 leukemic cells. The effect was rapid (<30 s), reached maximum at optimal chemotactic concentrations, and was completely blocked by pretreatment of monocytes with Bordetella pertussis toxin. A specific antiserum and heat inactivation blocked the induction of arachidonic release by MCP-1. No [3H]arachidonic acid release was observed in the absence of Ca2+ influx (5 mM EGTA or 5 mM Ni2+) or in monocytes loaded with a Ca(2+)-buffering agent. However, using ionophore-permeabilized monocytes and controlled intracellular Ca2+ concentration it was possible to dissociate MCP-1-induced Ca2+ influx from [3H]arachidonic acid release. Thus, the MCP-1-induced increase in [Ca2+]i is necessary but not sufficient for arachidonic acid accumulation. Phospholipase A2 inhibitors (mepacrine, p-bromophenacyl bromide, and manoalide) blocked monocyte polarization and chemotaxis induced by MCP-1. The related Cys-Cys chemokines RANTES and LD78/MIP1 alpha also induced a rapid release of [3H]arachidonic acid, and their chemotactic activity was blocked by phospholipase A2 inhibitors. Brief (5 min) pretreatment of monocytes with platelet-activating factor amplified MCP-1-induced arachidonic acid release and, at MCP-1 suboptimal concentrations, synergized in inducing monocyte migration. Since MCP-1 and platelet-activating factor are induced concomitantly by inflammatory cytokines in monocytes and endothelial cells, we speculate that the observed synergism may have in vivo relevance. The results presented here show that the Cys-Cys chemokines MCP-1, LD78/MIP1 alpha, and RANTES cause rapid release of arachidonic acid in monocytes and that this may be important in inducing monocyte chemotaxis.

The present study was aimed at defining the activity of human and mouse monocyte chemotactic protein-1 (MCP-1) on homologous and heterologous mononuclear phagocytes. Human natural and recombinant MCP-1 and mouse natural MCP-1/JE were tested as chemoattractants on human blood monocytes and mouse peritoneal macrophages. The human and murine cytokines were equiactive on human monocytes. Human MCP-1 was active on mouse macrophages but the maximal chemotactic effect elicited was about half that of human cells or of mouse MCP-1/JE or of reference chemoattractants. Human MCP-3, a recently identified member of the C-C chemokine family, with high sequence similarity to MCP-1/JE, was also active on mouse mononuclear phagocytes, though less so than mouse MCP-1/JE. These results caution against under-estimating the potential of MCPs when the human chemokines are applied in mice.

The human cytokine interferon-inducible protein 10 (IP-10) is a small glycoprotein secreted by activated T cells, monocytes, endothelial cells, and keratinocytes, and is structurally related to a family of chemotactic cytokines called chemokines. Although this protein is present in sites of delayed-type hypersensitivity reactions and lepromatous leprosy lesions, the biological activity of IP-10 remains unknown. We report here that recombinant human IP-10 stimulated significant in vitro chemotaxis of human peripheral blood monocytes but not neutrophils. Recombinant human IP-10 also stimulated chemotaxis of stimulated, but not unstimulated, human peripheral blood T lymphocytes. Phenotypic analysis of the stimulated T cell population responsive to IP-10 demonstrated that stimulated CD4+ and CD29+ T cells migrated in response to IP-10. This resembles the biological activity of the previously described T cell chemoattractant RANTES. Using an endothelial cell adhesion assay, we demonstrated that stimulated T cells pretreated with optimal doses of IP-10 exhibited a greatly enhanced ability to bind to an interleukin 1-treated endothelial cell monolayer. These results demonstrate that the IP-10 gene encodes for an inflammatory mediator that specifically stimulates the directional migration of T cells and monocytes as well as potentiates T cell adhesion to endothelium.