A quantitative microassay for leukocyte chemotaxis,using a microscopic slide system with complement-activating yeast particles as gradient source

A simple quantitative microassay was developed for studying polymorphonuclear leukocyte (PMNL) chemotaxis under conditions where the number of available cells is a limiting factor, e.g., pustules, neutropenia, small children and cerebrospinal fluid.PMNL suspensions are placed on glass slides to which fluorescein-labeled yeast particles have been fixed. After adherence, normal human serum is added to the slides. Owing to complement activation, a chemotactic gradient which attracts the adherent PMNL is formed around the yeast particles. The number of PMNL-associated yeast particles in the presence of normal serum is scored, and compared with cells migrating in the presence of inactivated serum or in the absence of serum. A locomotory index is calculated as the number of yeast particles associated with PMNL divided by the total number of yeast particles.     

Measurement of chemoattractant concentration profiles and diffusion coefficient in agarose

Migration of leukocytes is influenced by both the concentration and gradient of a chemoattractant. In the under-agarose assay, attractant diffuses through the medium away from the attractant well. For complete analysis of assay data, the diffusion coefficient of the attractant in the assay system must be known, and the attractant concentration profiles must be mathematically predictable. This paper describes a method whereby the diffusion coefficient of a chemoattractant through agarose may be determined, by measurement of attractant concentration profiles. The diffusion coefficient of FNLLP was found to be D = 1.0 X 10(-5) cm2/s at 37 degrees C. In addition it was found that the concentration profiles can be satisfactorily predicted by solution of the diffusion equation with appropriate boundary conditions.     

Effects of dialyzable leukocyte extracts (DLEs) with transfer factor activity on leukocyte migration in vitro. III. Characterization of the antigen-independent migration inhibition factor in DLEs as a neutrophil immobilizing factor.

In earlier evaluations of the agarose LMI assay as an in vitro test for studying the nature and mechanism of action of TF, we reported the existence of a component in human DLEs which caused noncytotoxic inhibition of the random migration of human PMNs. The LMI was not dependent on the stimulation of viable mononuclear leukocytes by antigen or mitogen to effect the release of mediators of cellular immunity such as LIF; rather, the LMI was promoted by the direct action of a preexisting component in DLEs on PMNs. We now present evidence that this "antigen-independent" LMI activity in DLE'S is similar to a NIF shown previously by Goetzl and co-workers to be present in acid extracts of leukocytes and to be released by phagocytosing PMNs. The comparison is drawn from several parameters: (1) cellular origin, (2) molecular weight, (3) target cell, (4) susceptibility to inactivation by heating or by incubation with pronase, trypsin, or chymotrypsin, and (5) ability to cause noncytotoxic inhibition of random migration or chemotaxis of PMNs.     

Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination

A method for quantitating the number of polymorphonuclear leukocytes (PMNs) adhering to endothelial cells in vitro is presented. Confluent human umbilical vein endothelial cells cultured in 24-well multiplates and treated with tumor necrosis factor (TNF ) were incubated subsequently wioth PMNs which adhere to the endothelial cell as a function of TNF concentration. Adherent PMNs and endothelial cells were proteolytically dissociated from the multiwell and, using an electronic particle counter, the number of endothelial cells and PMNs were determined simultaneously on the basis of size. The average number of PMNs adhering per endothelial cell was then calculated. The method is rapid and precise and offers an alternative to both tedious microscopic counting and the hazardous radiolabeling of PMNs.     

Effect of synthetic double-stranded polynucleotides on leukocyte migration

The effect of synthetic double-stranded polynucleotides (polyI:polyC) on migration of peripheral blood leukocytes from donors and patients with rheumatic fever was investigated. PolyI:polyC in large quantities was found not to inhibit migration of the donors' leukocytes, but it inhibited migration of leukocytes from patients with rheumatic fever, in whom antibodies against double-stranded RNA were found more often than in donors.Department of Internal Medicine with Rheumatological Research Laboratory, Volgograd Medical Institute. Laboratory of Radiation Biochemistry, Obninsk Institute of Medical Radiology, Academy of Medical Sciences of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 10, pp. 90–92, October, 1975.     

Chemotaxis of polymorphonuclear leukocytes to varicella-zoster virus antigens

Chemotaxis of polymorphonuclear leukocytes (PMNs) to various varicella-zoster virus (VZV) antigens was studied using a membrane filter method. Chemotactic activity of PMNs was detected in the presence of sonicated VZV antigen and soluble VZV skin test antigen. This activity was reduced when sonicated VZV antigen was treated with human seropositive serum or murine monoclonal antibodies which reacted with glycoprotein (GP) I or GP II of VZV. However, chemotaxis of PMNs was not reduced when sonicated VZV antigen was treated with human seronegative serum or a murine monoclonal antibody which reacted with GP IV. These results suggest that GP I and GP II act as chemoattractants to PMNs, and this mechanism might contribute to the resolution of the skin lesions of varicella and herpes zoster.     

A new assay for leukocyte chemotaxis using cell retrieval,electronic particle counting and flow cytometry

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.     

Binding and internalization of herpes simplex virus-antibody complexes by polymorphonuclear leukocytes

We studied the interactions between rabbit polymorphonuclear leukocytes (PMN) and the RE strain of herpes simplex virus type 1 (HSV-1) to determine better the role of inflammatory cells in herpetic stromal keratitis. PMN were found to be nonpermissive for HSV replication and were unable to bind virus in the absence of antibody. However, PMN did bind and internalize HSV-antibody complexes in vitro as was demonstrated visually by electron microscopic studies and quantitatively by measurement of activity associated with radiolabeled HSV-antibody complexes. Virus used for immune complex formation was labeled with either 125Iodine or 35S-methionine. In some experiments, anti-HSV IgG used for immune complex formation was labeled with 125Iodine before incubation with virus. Use of all three radiolabeling approaches resulted in the same general pattern of binding, indicating a requirement for both antibody and virus for interaction with PMN. The activity associated with PMN was increased by preincubation with complement. The results suggest an active role for PMN in controlling HSV infection through their ability to bind and ingest virus-antibody complexes.     

Integrin modulation and signaling in leukocyte adhesion and migration

Summary:  The movement of leukocytes from the blood into peripheral tissues plays a key role in immunity as well as chronic inflammatory and autoimmune diseases. The shear force of blood flow presents special challenges to leukocytes as they establish adhesion on the vascular endothelium and migrate into the underlying tissues. Integrins are a family of cell adhesion and signaling molecules, whose function can be regulated to meet these challenges. The affinity of integrins for their vascular ligands can be stimulated in subseconds by chemoattractant signaling. This aids in inducing leukocyte adhesion under flow conditions. Further, linkage of these integrins to the actin cytoskeleton also helps to establish adhesion to the endothelium under flow conditions. In the case of α4β1 integrins, this linkage of the integrin to the cytoskeleton is mediated in part by the binding of paxillin to the α4 integrin subunit and the subsequent binding of paxillin to the cytoskeleton molecule talin. The movement of leukocytes along the vascular endothelium and in between endothelial cells requires the temporal and spatial regulation of small guanosine triphosphatases, such as Rac1. We describe mechanisms through which α4β1 integrin signaling regulates appropriate Rac activation to drive leukocyte migration.     

Human polymorphonuclear neutrophils express RANK and are activated by its ligand, RANKL

The receptor activator of NF-κB (RANK) is especially well studied in the context of bone remodeling, and RANK and its ligand, RANKL, are key molecules in the induction of bone resorbing osteoclasts. We now report that polymorphonuclear neutrophils (PMNs) contain preformed RANK, stored in secretory vesicles and in specific granules. Upon stimulation of PMNs in vitro, RANK was translocated to the cell membrane. In patients with persistent bacterial infections, RANK surface expression was enhanced compared with that of healthy individuals. The functional activity of RANK was assessed by determining migration of PMNs toward RANKL. A time- and dose-dependent migration was seen, leading to the conclusion that RANK on PMNs is functional. We presume that regulated RANK expression contributes to the fine tuning of PMN migration, for example, on and through inflamed endothelium that is known to express RANKL.     

Functional insights on the polarized redistribution of leukocyte integrins and their ligands during leukocyte migration and immune interactions

Summary:  Cell–cell and cell–matrix interactions are of critical importance in immunobiology. Leukocytes make extensive use of a specialized repertoire of receptors to mediate such processes. Among these receptors, integrins are known to be of crucial importance. This review deals with the central role of integrins and their counterreceptors during the establishment of leukocyte–endothelium contacts, interstitial migration, and final encounter with antigen-presenting cells to develop an appropriate immune response. Particularly, we have addressed the molecular events occurring during these sequential processes, leading to the dynamic subcellular redistribution of adhesion receptors and the reorganization of the actin cytoskeleton, which is reflected in changes in cytoarchitecture, including leukocyte polarization, endothelial docking structure formation, or immune synapse organization. The roles of signaling and structural actin cytoskeleton-associated proteins and organized membrane microdomains in the regulation of receptor adhesiveness are also discussed.     

Correlation between leukocyte migration activity and RNA and DNA synthesis in experiments in vitro

In a parallel study of leukocyte migration in vitro in the presence of specific antigen and spontaneous synthesis of RNA and DNA the following significant correlations were found in lymphocytes in culture: 1) direct correlation between RNA and DNA synthesis; 2) close correlation between antigen-induced migration and the levels of RNA and DNA synthesis. The effect of the antigen was manifested as inhibition or stimulation of leukocyte migration. A high ratio between RNA synthesis and DNA synthesis corresponded to inhibition of migration, a low ratio to its stimulation. The value of the ratio itself varied mainly on account of changes in the level of DNA synthesis. The participation of T and B lymphocytes in the regulation of leukocyte mobility under the influence of antigen is discussed.Department of Microbiology and Immunology, Institute of Experimental Medicine, Academy of Medical Sciences of the USSR. Department of Propedeutics of Children's Diseases, Pediatric Medical Institute, Ministry of Health of the RSFSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. I. Ioffe.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 721–723, June, 1978.     

An objective filter-based, enzymatic method for the in vivo measurement of the migration of human polymorphonuclear leucocytes

Incorporation of control valves into a previously described device enabled us to regulate the formation of 8 suction blisters on the upper surface of the forearm in adult human volunteers. After the removal of the raised epidermis and blister fluid, uniform areas of denuded dermis were obtained by placing hollow adhesive ring reinforcers onto each of the regions of exposed dermis. Single or double nitrocellulose filters were then placed onto each of the areas of moistened, exposed dermis. The chemotactic tripeptide FMLP was incorporated into 1% agarose containing 0.1% bovine serum albumin (BSA) to give a concentration range of 10(-8) M to 10(-6) M FMLP. In control systems the FMLP was omitted. Cylindrical agarose blocks +/- FMLP were then placed onto the filters and encased in individual perspex cups glued firmly onto the skin. The filter(s) and agarose blocks were replaced at 2 h intervals and polymorphonuclear leucocyte (PMNL) migration onto (single filter) and into (double filter) the filters was measured by microscopic enumeration or according to the amount of myeloperoxidase (MPO) and lysozyme in the supernatants of filters immersed in 0.1% Triton-X for 10 min to lyse the PMNL. Microscopic enumeration was found to be unsuitable but the method based on MPO and lysozyme release from filter-associated PMNL was rapid, accurate and reproducible. Detectable PMNL migration (greater than 90%) occurred at 3-4 h and was maximal at 8-10 h. This pattern was observed for both the control and FMLP-containing systems. However, PMNL migration was significantly greater in FMLP-exposed dermis. FMLP at 10(-6) M was found to promote maximal PMNL migration. Significantly greater MPO and lysozyme activities were observed with the double filter system. This method is suitable for the objective quantitation of PMNL migration in vivo.     

Defective monokine production and decreased responsiveness of polymorphonuclear leukocytes to recombinant interleukin-1 in asthmatic patients

Augmented IgE production and increased infections are often seen in allergic patients. As monocytes (MN) and polymorphonuclear leukocytes (PMN) are involved in both immune regulation and inflammatory reaction, MN function in terms of monokine production stimulated with lipopolysaccharide (MN supernatant; MN-sup) and its biological activity and the response of PMN to MN-sup and recombinant interleukin-1 (rIL-1) regarding chemotactic activity and expression of IgG Fc receptor (FcR) were studied in 26 normal children and 28 new and 22 hyposensitized (HS) asthmatic children. The results showed the following. (1) There was no difference in IL-1 production, as assayed by thymocyte proliferation, among the three groups. (2) All MN-sup from the three groups could enhance IL-2 production, but that of new patients was less efficient. (3) In the absence of PWM, MN-sup of new patients greatly augmented the production of IgG, IgA, IgM, and IgE, but that of HS patients could enhance only IgE synthesis. (4) MN-sup of patients enhanced less efficiently the chemotactic activity and FcR expression of PMN from healthy volunteers, and PMN from asthmatics responded much less vigorously to rIL-1 regarding the above-mentioned functions. (5) The number of PMN with membrane IL-1 was much lower in allergic patients. Thus the abnormal MN and PMN functions may be used to explain partly the augmented IgE production and increased infections in allergic patients.     

金黄色葡萄球菌对中性粒细胞粘附特性的影响

目的：研究中性粒细胞在外源病原微生物金黄色葡萄球菌及其分泌物 免疫激活状态下的粘附特性,考察不同的细菌浓度对中性粒细胞粘附性的影响。材料与方法：采用微管吸吮技术测量大鼠中性粒细胞经不同浓度金黄色葡萄菌及其分泌物刺激后与内皮细胞的粘附特性。结果：中性粒细胞经金黄色葡萄球菌免疫激活后与内皮细胞的粘附力和对照组比较均显著增高,但随着细菌浓度的提高而表现出饱和效应。用金黄色葡萄球菌分泌物（培养液）处理中性粒细胞中,其与内皮细胞的粘附力和对照组比较却有所降低。将经细菌处理后的中性粒细胞分离出再与内皮细胞黏附,黏附力增加幅度更大。结论：中性粒细胞经金黄色葡萄球菌免疫激活后粘附分子表达增加,但金黄色葡萄球菌分泌物可能对中性粒细胞粘附分子表达起抑制作用。在此过程中,内皮细胞可能受到金黄色葡萄球菌及其分泌物的损伤。     

Quantitation of rubella virus by competitive enzyme immunosorbent assay

A simple and rapid technique for quantification of rubella virus is described. The specificity of the competitive enzyme linked immunosorbent assay (ELISA) was quantified by comparing the slopes and the Y intercepts of the curves obtained when viral antigen vs. control antigen was used. The curves were derived by plotting the absorbance ratio against the logarithm of the antigen concentration. The technique was reproducible, its sensitivity depending on the purity of the antigen used. In ELISA, when an antigen precipitated by ammonium sulfate was used, the sensitivity expressed in protein/ml was 7 micrograms and when an antigen purified by sucrose gradient was used, 70 ng, whereas the limit of sensitivity in the standard technique of hemagglutination was only 38 micrograms.     

The effect of chemotaxis and chemokinesis on leukocyte locomotion: A new interpretation of experimental results

A mathematical model is developed to describe the motion ofleukocytes through a Boyden chamber. The model is based on theKeller–Segel model of cell motion and comprises threepartial differential equations which describe the evolutionof the neutrophils, the chemoat-tractant, and a neutrophil-derivedchemokinetic factor. Where other authors have concentrated onchemotaxis, here attention is focused on the manner in whichthe chemokinetic factor influences neutrophil locomotion. Numericalsimulations show how the number of neutrophils initially placedon top of the chamber affects cellular motion through the systemand reproduce the qualitative behaviour observed by Takeuchi& Persellin (Am. J. Physiol. 236, C22–C29). In particular,the simulations show how dense packing of the neutrophils increasesthe levels of the chemokinetic factor. This enhances randomcell motion and increases the speed with which the neutrophilsreach the source of chemoattractant. For a particular asymptoticlimit of the system parameters, the model reduces to a nonlinearpartial differential equation for the neutrophils. Similaritysolutions of this caricature model yield algebraic expressionsrelating the speed with which the neutrophil front penetratesinto the chamber to the number of neutrophils initially placedon top of it. The implications of the results are also discussed.     

Quantitation by myeloperoxidase assay of neutrophil accumulation at the site ofin vivo allergic reactions

Skin window techniques to investigate neutrophil inflammatory reactions in human skin have been limited by cellular distortion and difficulties in quantitation. We have developed a quantitative approach based on the assessment of the myeloperoxidase (MPO) released from sonicated membrane filters to which exuding inflammative cells had adhered. Our studies have shown that (1) a standard curve can be derived for each subject from the linear relationship between amounts of MPO released and designated numbers of blood neutrophils which have been aspirated onto such filters; (2) the number of neutrophils adhering to filters appended to skin window sites can then be reliably estimated by comparing the amount of MPO released by sonication of such filters with the standard curve; and (3) neutrophil accumulation is greater at pollen-challenged than control sites in sensitive subjects.