Binding of protein chemotactic factors to the surfaces of neutrophil leukocytes and its modification with lipid-specific bacterial toxins

Summary The binding to neutrophil leukocytes of human serum albumin (HSA), which is chemokinetic for leukocytes, i.e. influences their rate of locomotion, and of alkali-denatured HSA, which is chemotactic for leukocytes, i.e. influences their direction of locomotion, was studied. Native serum albumin showed low affinity binding to the neutrophil surface. Denatured serum albumin showed saturable binding with a K_a of approximately 10⁶ litres per mole to about 10⁶ binding sites per cell. Another protein chemotactic factor, _s-casein, gave similar binding. These results exclude that chemotactic reactions to denatured proteins are mediated in a completely non-specific manner and suggest the presence on the cell of a restricted number of defined recognition sites. Binding was reduced following treatment of the cells with either of two lipid-specific bacterial toxins, perfringolysin, the -toxin of Clostridium perfringens, an oxygen-labile cholesterol-specific toxin, and Staphylococcus aureus Sphingomyelinase C. Both have previously been shown to reduce chemotactic reactions and both were used at doses which did not reduce cell viability. These results suggest an important, and possibly direct, role for membrane lipid in the binding sites for chemotactic factors. Visual analysis of the behaviour of perfringolysin-treated neutrophils showed that these cells were still capable of chemotactic locomotion. The cells appeared to be less efficient than normal in detecting chemotactic gradients only when at a distance from the gradient source, a finding which is consistent with reduced binding of the chemotactic factor to the cell surface.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

The investigation of the ultrastructural neutrophil changes in alloxan-induced diabetes in rats: response to a chemotactic challenge

Experimental diabetes is one of the most popular conditions in which to study the relation between neutrophil leukocyte activity and periodontal destruction. The aetiology of neutrophil dysfunction in the gingival tissue associated with diabetes has yet to be clarified. Diabetes in rats decreases neutrophil chemotactic activity in proportion to the severity of this systemic disorder. The present study was carried out to evaluate the relationship between the severity of diabetes and the neutrophil response to two chemotactic agents, and to correlate the observed neutrophil defects with the degree of diabetes. In this study two chemotactic agents, casein (0.2 μl, 2 mg ml^?1) or N‐formylmethionylleucylphenylalanine (FMLP; 0.2 μl, 10^?4 M ), were placed into the gingival crevices of alloxan‐induced diabetic rats. Gingival biopsies were taken 15 min later and then at 5‐min intervals up to 45 min and investigated by electron microscopy. Adherence and migration were observed in the rats with moderate diabetes 30 min after the application of casein. There was chemotaxis after 35 min of administration of the peptide FMLP. By 40 min neutrophils with pyknotic nuclei were observed. At 45 min neutrophils with a decreased number of granules were present. As the severity of the diabetes increased, the neutrophils degenerated and were structurally distorted. In the rats which had alloxan‐induced diabetes there was abnormal periodontal damage. This damage is thought to be related to dysfunctional neutrophils. These findings many contribute to an answer to the following question: why is there an apparent variability in the susceptibilty of periodontal breakdown in diabetics? Copyright © 2003 John Wiley & Sons, Ltd.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Composition and size distribution of bovine casein micelles

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, κ-casein varied from 7.7 to 11.4% of total casein, giving α_s/κ ratios of 6.1, 4.7 and 3.3.A plot of κ-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between κ-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a pre-dominant surface location for κ-casein, whether the micelles are natural or artificial and whether they are aggregated or by Ca²⁺ alone oy Ca²⁺ together with calcium phosphate-citrate complex.     

Dual effects of formylpeptides on the adhesion of endotoxin-primed human neutrophils

Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (> 5 × 10^?8 M ) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10^?10 M to 5 × 10^?9 M ) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (> 10^?7 M ) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (> 10^?9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10^?9 M ) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10^?9 M ) and high (5 × 10^?7 M ) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.     

Conformation of αs-Casein in Various Concentrations

Conformation of α_s-casein and its association were investigated from behaviors of tyrosyl and tryptophyl residues and hydrophobic sites. The chromophoric residues and ANS binding sites were buried into a region inaccessible to solvent with increasing concentration of α_s-casein. It is considered that the association of α_s-casein with concentration is proceeded by the hydrophobic sites to be able to bind ANS and the hydrophobic segments in which tyrosyl and tryptophyl residues exist. Below 0.04% of α_s-casein, α_s-casein exists in the monomer state and 80% of tyrosyl and tryptophyl residues are accessible to aqueous solvent. The hydro-phobic sites of α_s-casein may be exposed to solvent in the monomer state.     

Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium

Monocyte emigration into areas of inflammation is initiated by monocyte adherence to the microvascular endothelium which may be induced by the local production of chemotactic factors at the inflammatory site. However, it is not clear whether such stimuli act on the monocyte and/or the endothelial cell to promote this effect. Accordingly, the effect of the chemotactic peptides C5a des arg and formyl-methionyl-leucyl-phenylalanine (FMLP) on human monocyte adherence to human microvascular endothelial cell monolayers was investigated in vitro. Monocytes (92 to 98% pure) were isolated by discontinuous plasma-Percoll density gradients and cell elutriation, methods designed to minimize monocyte exposure to endotoxin. Mean spontaneous (unstimulated) adherence of 111Indium-tropolonate-radiolabeled monocytes to microvascular endothelial cell monolayers was 19.7% +/- 1.3. Monocyte adherence to microvascular endothelial cell monolayers was stimulated in a dose-response fashion in the presence of C5a des arg or FMLP to a maximum mean adherence of 47.2% +/- 2.9 or 43.8% +/- 2.2, respectively. C5a des arg or FMLP stimulated monocytes to adhere to monolayers of human vascular smooth muscle cells, human dermal fibroblasts, or serum-coated plastic wells in a comparable fashion as to endothelial cells. The simultaneous presence of both chemotactic peptides C5a des arg and FMLP in the assay system stimulated monocyte adherence to the same degree as either stimulus alone. This finding suggested that those monocytes stimulated to adhere by C5a des arg were the same subpopulation responding to FMLP. Spontaneous monocyte adherence (in the absence of chemotactic peptides) to both endothelial cell monolayers and serum-coated plastic wells was reduced in the presence of plasma, but chemotactic peptides induced a significant, albeit reduced, adhesion of monocytes in this circumstance. The pretreatment of monocytes with either C5a des arg or FMLP prior to the adherence assay induced stimulus-specific desensitization of monocyte adherence. Neither a desensitization nor stimulated monocyte adherence occurred when endothelial cell monolayers or serum-coated plastic wells were pretreated with either of the chemotactic peptides. The fixation of endothelial cell monolayers prior to the adherence assay did not alter the degree of spontaneous, C5a des arg-stimulated, or FMLP-stimulated monocyte adherence. These data suggest that the stimulated adhesion of monocytes to endothelial cells by C5a des arg or FMLP represents primarily an effect of these chemotactic peptides on the monocyte.     

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system¹. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury^2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes⁴. This method focuses on neutrophil transmigration across human endothelial cells.One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)⁵. Neutrophil isolation has been described visually elsewhere⁶; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression⁷. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature^7,8.The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro^9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.     

Structure of Bovine αs-Casein

The secondary structure of bovine α_s-casein and chemically modified α_s-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of α_s-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-α_s-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-α_s-casein had two infrared absorption peaks centered at 1625 and 1643 cm^?1 in D₂O-CH₃OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH₃OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-α_s-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified α_s-caseins.     

Calcium-induced precipitation of αs1-casein: effect of modification of lysine residues

Turbidity measurements have been used to study the calcium-induced precipitation of α₃₁-casein whose amino groups have been modified by reaction with dansyl chloride and fluorescamine. Provided account is taken of the change in charge wrought by these modifications, the precipitation behaviour of these modified caseins is shown to be no different from that of the native α_s1-casein protein. The results provide further support for the previously suggested isoelectric precipitation model for this reaction.     

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I_e. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 μM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.     

Decreased CXCR1 and CXCR2 expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration

Introduction

In anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.

Methods

Membrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.

Results

Expression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.

Conclusions

Expression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).     

Temporal adaptation of neutrophil oxidative responsiveness to n-formyl-methionyl-leucyl-phenylalanine. Acceleration by granulocyte-macrophage colony stimulating factor

This investigation was undertaken to clarify the mechanism by which purified recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) potentiates neutrophil oxidative responses triggered by the chemotactic peptide, FMLP. Previous studies have shown that GM-CSF priming of neutrophil responses to FMLP is induced relatively slowly, requiring 90 to 120 min of incubation in vitro, is not associated with increased levels of cytoplasmic free Ca2+, but is associated with up-regulation of cell-surface FMLP receptors. We have confirmed these findings and further characterized the process of GM-CSF priming. We found that the effect of GM-CSF on neutrophil oxidative responsiveness was induced in a temperature-dependent manner and was not reversed when the cells were washed extensively to remove the growth factor before stimulation with FMLP. Extracellular Ca2+ was not required for functional enhancement by GM-CSF and GM-CSF alone effected no detectable alteration in the 32P-labeled phospholipid content of neutrophils during incubation in vitro. Our data indicate that GM-CSF exerts its influence on neutrophils by accelerating a process that occurs spontaneously and results in up-regulation of both cell-surface FMLP receptors and oxidative responsiveness to FMLP. Thus, the results demonstrate that, with respect to oxidative activation, circulating endstage polymorphonuclear leukocytes are nonresponsive or hyporesponsive to FMLP; functional responsiveness increases dramatically as surface FMLP receptors are gradually deployed after the cells leave the circulation. Thus, as neutrophils mature, their responsiveness to FMLP changes in a manner which may be crucial for efficient host defense. At 37 degrees C, this process is markedly potentiated by GM-CSF. We conclude that endogenous GM-CSF, released systemically or at sites of infection and inflammation, potentially plays an important role in host defense by accelerating functional maturation of responding polymorphonuclear leukocytes.     

Adhesion Between Human Neutrophils and Immobilized Endothelial Ligand Vascular Cell Adhesion Molecule 1: Divalent Ion Effects

Integrin-mediated adhesion of circulating neutrophils to endothelium during inflammation involves multiple adhesion molecules on both neutrophils and endothelium. Most studies of neutrophil adhesion have focused on adhesion to ICAM-1 (mediated by β₂ integrins), but interaction with the endothelial ligand vascular cell adhesion molecule 1 (VCAM-1) may also play a role in neutrophil adhesion to activated endothelium. In this study we demonstrate significant adhesion between neutrophils and VCAM-1 mediated by β₁ integrins, principally via α₄β₁ (VLA-4). We characterize the dynamics of adhesion in terms of rate constants for a two-step bond formation process, the first involving juxtaposition of active molecules with substrate and the second involving bond formation. The results indicate that the first step is rate limiting for VLA-4-VCAM-1 interactions. Changing divalent cation composition affects these coefficients, implicating molecular conformational changes as a key step in the process.