Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion.

The receptor-mediated adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This paper describes a calculational method which simulates the interaction of a single cell with a ligand-coated surface under flow. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the resulting receptor-ligand springs, the response of springs to strain, and the magnitude of the bulk hydrodynamic stresses. The model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the method can generate meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the strain of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same strain. Our analysis of neutrophil adhesive behavior on selectin-coated (CD62-coated) surfaces in viscous shear flow reported by Lawrence and Springer (Lawrence, M.B., and T.A. Springer 1991. Cell. 65:859-874) shows the fractional spring slippage of the CD62-LECAM-1 bond is likely below 0.01. We conclude the unique ability of this selectin bond to cause neutrophil rolling under flow is a result of its unique response to strain. Furthermore, our model can successfully recreate data on neutrophil rolling as function of CD62 surface density.     

Tumour necrosis factor-alpha and interleukin-8 inhibit neutrophil migration in vitro and in vivo

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-alpha) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-alpha (mrTNF-alpha) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-alpha into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.     

The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo

The third member of the family of junctional adhesion molecules (JAMs), JAM-3, also called JAM-C, was recently shown to be a novel counter-receptor on platelets for the leukocyte beta(2)-integrin Mac-1 (alphaMbeta(2), CD11b/CD18). Here, new functional aspects of the role of endothelial cell JAM-C were investigated. Endothelial cells express JAM-C, which is predominantly localized within junctions at interendothelial contacts, since it codistributes with a tight junction component, zonula occludens-1. Whereas JAM-C does not participate in neutrophil adhesion to endothelial cells, it mediates neutrophil transmigration in a Mac-1-dependent manner. In particular, inhibition of JAM-C significantly reduced neutrophil transendothelial migration, and the combination of JAM-C and platelet/endothelial cell adhesion molecule-1 blockade almost completely abolished neutrophil transendothelial migration in vitro. In vivo, inhibition of JAM-C with soluble mouse JAM-C resulted in a 50% reduction of neutrophil emigration in the mouse model of acute thioglycollate-induced peritonitis. Thus, JAM-C participates in neutrophil transmigration and thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies.     

Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (greater than 90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Peptides in the hemolymph of Hermetia illucens larvae completely inhibit the growth of Klebsiella pneumonia in vitro and in vivo

Extensive use of antibiotics has caused the microbial resistance to rise drastically within the last few decades, and new approaches are therefore needed to develop effective antibacterial substances. In this study, we identified peptide in the hemolymph of Hermetia illucens larvae using reverse-phase chromatography, HPLC and Nano-LC-ESI-MS/MS system. We investigated the antibacterial effect of HP/F9 peptides against Klebsiella pneumoniae in vitro and in vivo. The peptide effectively inhibited the growth of K. pneumoniae in vitro and completely removed K. pneumoniae from the lungs of mice. Importantly, peptides (22,000 Da, HP/F9) successfully reduced lung inflammation upon K. pneumoniae infection. These results indicate that the HP/F9 peptide from H. illucens larva can effectively protect the mouse from K. pneumoniae infection. HP/F9 could be a new candidate for the development of effective antibacterial substance.     

Peptides from Paracoccidioides brasiliensis GP43 inhibit macrophage functions and inflammatory response

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. Its major antigen is a 43-kDa glycoprotein. Gp43 embodies different functions: it participates in evasion mechanisms during the installation of primary infection, stimulates granuloma-like formation in vitro and presents T-cell epitopes that induce protective response against the fungus. Here, we investigated epitopes from gp43 inhibitory of both, macrophage functions and inflammatory reaction. Different gp43 peptides, spanning the entire sequence of the molecule, were added to cultures of bone marrow-derived macrophages. After challenge with zymosan or Pb cells, phagocytic indexes were measured. Peptides expressed on the molecule surface were determined by graphic analysis using the Protean module; DNAstar Inc. Two peptides which decreased phagocytic index and were expressed at the surface of the molecule, P4 and P23, were selected for further studies. It was shown that both inhibited the release of NO by zymosan stimulated macrophages while enhanced release of H₂O₂. The release of TNF-α in culture supernatants from in vitro phagocytic tests showed different response depending of P4 concentration (data not shown). In vivo assays with Mycobacterium bovis – bacillus Calmette–Guérin (BCG) or Pb cells demonstrated that these peptides presented non-specific and specific anti-inflammatory properties.     

Antibodies to the L1 adhesion molecule inhibit Schwann cell ensheathment of neurons in vitro

To investigate whether neural adhesion molecules are involved in neuron- induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.     

Perflubron attenuates neutrophil adhesion to activated endothelial cells in vitro

Infiltration of activated neutrophils into the lung appears to be a key element in the severe lung injury that develops in animal models of acute lung injury. Partial liquid ventilation with perflubron has been shown to ameliorate tissue damage compared with conventional mechanical ventilation in acute lung injury models. Pilot experiments indicated that indirect exposure to perflubron could modulate the degree to which subsequent neutrophil binding to endothelial cell monolayers was upregulated after lipopolysaccharide activation. Endothelial cell monolayers preexposed to perflubron showed >40% reductions in the surface steady-state levels of E-selectin and intercellular adhesion molecule-1 achieved after proinflammatory activation (P < 0.05), which correlated with a reduction in the real-time association constants measured by biosensor techniques. These results indicate that direct contact with the perflubron liquid phase is not necessary to attenuate inflammatory responses. Rather, diffusion of perflubron from the alveolar space into the adjacent pulmonary vascular endothelial layer may modulate neutrophil adhesion and thereby reduce the rate of infiltration of activated neutrophils into the injured lung.     

Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.     

Tumor promoter PMA enhances kindlin-2 and decreases vimentin recruitment into cell adhesion sites

Phorbol diester PMA (phorbol 12-myristate 13-acetate) is a well-known promoter of tumor progression. PMA also regulates cell adhesion by several mechanisms including conformational activation of integrins and integrin clustering. Here, PMA was shown to induce lamellipodia formation and reorganization of the adhesion sites as well as actin and vimentin filaments independently of integrin preactivation. To further analyze the mechanism of PMA action, the protein composition in the α1β1 integrin/collagen IV adhesion sites was analyzed by mass spectrometry and proteomics. In four independent experiments we observed the reduced recruitment of vimentin in relation to integrin α1 subunit. This was in full agreement with the fact that we also detected the retraction of vimentin from cell adhesions by confocal microscopy. Furthermore, the accumulation of kindlin-2 into cell adhesions was significantly increased after PMA treatment. Kindlin-2 siRNA inhibited cell spreading as well as the formation of actin fibrils and cell adhesions, but did not prevent the effect of PMA on lamellipodia formation. Thus, kindlin-2 recruitment was considered to be a consequence rather than the primary cause for the loss of connection between vimentin and the adhesion sites.     

Cannabinoids stimulate and inhibit testosterone production in vitro and in vivo

The major psychoactive Δ⁹-tetrahydrocannabinol (THC) and the non-psychoactive cannabinol (CBN) of cannabidiol (CBD) can both stimulate and inhibit testicular testosterone (T) production $\text{in vitro}$ and $\text{in vivo}$. At nanomolar concentrations, these cannabinoids stimulate T production by decapsulated mouse testes while, in micromolar amounts, the effects are markedly inhibitory.     

In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion

Trypanosoma cruzi causing Chagas' disease needs to invade host cells to complete its life cycle. Macromolecules on host cell surfaces such as laminin, thrombospondin, heparan sulfate, and fibronectin are believed to be important in mediating parasite-host cell adhesions and in the invasion process of the host cell by the parasite. The SELEX technique (systematic evolution of ligands by exponential enrichment) was used to evolve nuclease-resistant RNA ligands (aptamer = to fit) that bind with affinities of 40-400 nm to parasite receptors for the host cell matrix molecules laminin, fibronectin, thrombospondin, and heparan sulfate. After eight consecutive rounds of in vitro selection four classes of RNA aptamers based on structural similarities were isolated and sequenced. All members of each class shared a common sequence motif and competed with the respective host cell matrix molecule that was used for displacement during the selection procedure. RNA pools following seven and eight selection rounds as well as individual aptamers sharing consensus motifs were active in inhibiting invasion of LLC-MK(2) monkey kidney cells by T. cruzi in vitro.     

Canine neutrophil margination mediated by lectin adhesion molecule-1 in vitro.

The contributions of the canine neutrophil lectin adhesion molecule-1 (LECAM-1) (canine homologue of the murine MEL-14 Ag) in neutrophil-endothelial cell adhesion and transendothelial migration were studied using anti-LECAM-1 mAb, CL2/6, and SL1 under static conditions and at wall shear stresses of up to 1.85 dynes/cm2 (dpc). Both mAb were found to inhibit attachment of neutrophils to cytokine-stimulated canine jugular vein endothelium. The inhibitory effects of the anti-LECAM-1 mAb were more evident at a wall shear stress of 1.85 dpc (greater than 50%) than at 0.23 dpc or under static conditions (approximately 30%). In contrast the anti-CD18 mAb, R15.7, exhibited higher inhibitory ability at the lower shear stress and under static conditions with marginal inhibition of adhesion at 1.85 dpc. Anti-LECAM-1 and anti-CD18 mAb showed additive inhibitory effects at the lower wall shear stress and under static conditions. Chemotactic stimulation of the neutrophils caused rapid down-regulation of LECAM-1 from the neutrophil surface and reduced adhesion by 60% at a wall shear stress of 1.85 dpc. This inhibition was not additive to anti-LECAM-1 mAb. Pretreatment with CL2/6 or SL1 did not affect trans-endothelial migration of adherent neutrophils under any experimental conditions tested. Anti-CD18 mAb, however, blocked transendothelial migration by 98% and 56% under static condition and at a wall shear stress of 0.23 dpc, respectively. The results in this report indicate that canine LECAM-1 is involved in the initial adhesion of unstimulated neutrophils to cytokine-stimulated endothelial cells under flow, but in contrast to CD18-integrins, plays no role in the transendothelial migration.     

TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.     

Sensory neurons influence the expression of cell adhesion factors by cutaneous cells in vitro and in vivo

Dorsal root ganglion (DRG) neurons co-cultured with skin-derived fibroblast-like cells (FLCs) show a strong neurite outgrowth. However, when physical contact between FLCs and neurons is prevented with membrane inserts, the DRG neurons exhibit a low survival and a deficient neurite growth. This indicates that cell adhesion molecules influence neuronal survival and neurite growth in co-cultures. The aim of the present study is to find out if selected adhesion molecules are expressed by cultivated FLCs with and without nervous influences, and/or by normal and denervated whole skin. RT-PCR data show that cultured FLCs and denervated skin express L1, N-CAM, N-cadherin and ninjurin, but not neurofascin or TAG-1. However, cultured FLCs exposed to DRG homogenates and innervated skin express N-cadherin only. Following application of neutralizing L1-, N-cadherin- and ninjurin-antibodies (but not N-CAM-antibodies) in the culture medium the mean number of surviving neurons is decreased. Co-cultures incubated with L1-, N-cadherin- or ninjurin-antibodies all show significantly less neurite outgrowth compared to controls. In conclusion, the findings in this paper indicate (i) that FLCs cultured in vitro and denervated whole skin express the cell adhesion factors L1, N-CAM, N-cadherin and ninjurin, (ii) that FLCs treated with neural molecules and innervated whole skin express N-cadherin only, (iii) that L1, N-cadherin and ninjurin are important for DRG neurons co-cultured with FLCs in vitro in terms of survival and neurite extension and (iv) that there may exist subpopulations of DRG-neurons with different sensitivities for N-cadherin- and ninjurin-antibodies.     

Thiol-sensitive sites in cell adhesion. Decreased entry of SH-binding reagents into attached BHK cells.

Studies were carried out to learn more about the critical SH groups involved in cell spreading. Pretreatment of suspended baby hamster kidney (BHK) cells with 3 mM-iodoacetate or iodoacetamide for 10 min at 4 degrees C completely inhibited the ability of the cells to spread on fibronectin-coated substrata. If, however, BHK cells were permitted to attach and spread before being treated with the SH-binding reagents, and then harvested by trypsinization and assayed for spreading on fibronectin-coated substrata, there was no inhibition of cell spreading. The extent of prior attachment required before the cells became insensitive to the SH-binding reagents was tested and was found to occur early during the cell adhesion process, before any cell spreading was observed. In analytical experiments, there did not appear to be any difference in the total number of SH groups between suspended or spread cells as determined with 5,5'-dithiobis-(2-nitrobenzoic acid). The uptake of radiolabelled iodoacetate into intact spread cells, however, was found to be 3.5 times less than that found with suspended cells. On the other hand, the distribution of incorporated radioactivity into suspended and spread cells was similar. Most of the radioactivity (approximately 70%) was incorporated into small molecules (e.g. glutathione and cysteine), less (approximately 20%) was incorporated into cytoplasmic proteins, and the least incorporation (approximately 10%) was into the cell cytoskeleton. The data are interpreted to indicate there is a decreased permeability of spread cells to the SH-binding reagents.     

Chronic inflammatory disease alters adhesion molecule requirements for acute neutrophil emigration in mouse skin

Mutant mice triply deficient in ICAM-1, E-selectin, and P-selectin did not develop the neutrophilic skin lesions that spontaneously arise in mutants doubly deficient in E-selectin and P-selectin. Thus, ICAM-1 is essential to skin disease resulting from endothelial selectin deficiency. During experimental dermatitis, acute neutrophil emigration was completely prevented in young mice deficient in both selectins (E/P and E/P/I mutants). However, older E/P mutants with spontaneous skin lesions displayed an endothelial selectin-independent pathway for acute neutrophil emigration. In contrast, emigration remained compromised in E/P/I mutants and CD18 mutants regardless of age or lesions. Experimentally induced chronic lesions elicited this pathway for acute emigration in young E/P mutants. Thus, an endothelial selectin-independent pathway for acute neutrophil emigration is induced in E/P mice by chronic inflammation at distant sites, and this pathway may contribute to skin disease resulting from endothelial selectin deficiency.