The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis.

Interleukin 15 (IL-15) is a novel cytokine with interleukin-2-like activity. It is also a potent T-lymphocyte chemoattractant. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the presence of activated T lymphocytes, macrophages and synoviocytes in the synovial membrane. The mechanisms of T-cell activation in RA are currently unclear. We report the presence of high concentrations of IL-15 in rheumatoid arthritis (RA) synovial fluid and have demonstrated its expression in the synovial membrane lining layer by immunohistochemistry. RA synovial fluids were found to contain chemotactic activity, which was attributable in part to the presence of IL-15. Moreover, in a murine model, injection of recombinant IL-15 was found to induce a local tissue inflammatory infiltrate consisting predominantly of T lymphocytes. Synovial fluid T lymphocytes proliferate in response to IL-15, demonstrating that continued responsiveness to IL-15 is a feature of T cells after entry into the synovial compartment. These data suggest that IL-15 can recruit and activate T lymphocytes in the synovial membrane, thereby contributing to RA pathogenesis.

Chemoattraction of human blood T lymphocytes by interleukin-15.

Recombinant interleukin (IL)-15, derived from a simian kidney epithelial cell line, is a chemoattractant for human blood T lymphocytes judged by its ability to increase the proportion of cells in polarized morphology, to stimulate invasion of collagen gels containing IL-15, and to increase the proportion of locomotor cells observed by time-lapse videorecording. The ability of lymphocytes to respond was partly, but not completely, inhibited by pretreatment with anti-IL-2 receptor beta-chain. The activity of IL-15 was completely abolished by preincubation with aIL-15 but unaffected by preincubation with aIL-2. No response of monocytes, neutrophils, or B lymphocytes to IL-15 was observed.

Antigen-induced locomotor responses in lymphocytes.

The effect of protein antigens on the locomotion of lymphocytes from the lymph nodes draining the site of antigenic challenge in immunized mice, and from the same nodes in control mice, was studied in filters using a checkerboard assay in which the absolute concentration and the concentration gradient of attractant was varied in a series of chambers. Serum albumin (HSA or BSA) was chemokinetic for unimmunized lymphocytes inasmuch as the distance migrated into filters by cells in its presence varied with the absolute concentration of albumin, but not with the concentration gradient, indicating an influence of the serum albumin on the rate but not on the direction of locomotion. Ovalbumin and nonalbumin proteins did not show this effect. Using the same assay, the migration of primed lymphocytes in the presence of the priming antigen was shown to be influenced by the antigen gradient in a way that suggested a positive chemotactic response of the lymphocytes to antigen. This response was only shown clearly when the cells were in a chemokinetic medium containing serum albumin.

Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices.

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.

Chemokinetic accumulation of human neutrophils on immune complex-coated substrata: analysis at a boundary.

The locomotory behavior of human blood neutrophil leukocytes was studied at a boundary between two surfaces with different chemokinetic properties. This was achieved by time-lapse cinematography of neutrophils moving on coverslips coated with BSA, then part-coated with immune complexes by adding anti-BSA IgG with a straight-line boundary between the BSA and the immune complexes. Cell locomotion was filmed in microscopic fields bisected by the boundary, and kinetic behavior was assessed by comparing speed (orthokinesis), turning behavior (klinokinesis), and the rate of diffusion of the cells on each side of the boundary, using a recently described mathematical analysis of kinesis. In the absence of serum or complement, the proportion of motile cells and their speed and rate of diffusion were greater on BSA than on antiBSA, but there was no consistent difference in turning behavior between cells on the two surfaces. The immune complexes were therefore negatively chemokinetic in comparison with BSA, and this resulted from a negative orthokinesis with little or no contribution from klinokinesis. As would be predicted theoretically, this resulted in gradual accumulation of cells on the immune complexes even in the absence of a chemotactic factor. In further studies, a parallel plate flow chamber was used to show that, under conditions of flow, neutrophils accumulated much more rapidly on a surface coated with BSA-anti-BSA than on BSA alone. Moreover, neutrophils on immune complex-coated surfaces lost their ability to form rosettes with IgG-coated erythrocytes. This suggests that neutrophils on immune complex-coated surfaces redistribute their Fc receptors (RFc gamma) to the under surface, and that the lowered speed of locomotion is due to tethering of neutrophils by substratum-bound IgG-Fc.

A visual study of chemotaxis of human lymphocytes using a collagen-gel assay.

Time-lapse cinematography was used to study the chemotactic responsiveness of human blood lymphocytes as defined by morphological orientation and directional locomotion in gradients. At present, evidence for lymphocyte chemotaxis is indirect since neither of these essential features can be demonstrated with Boyden filter assays. Few lymphocytes direct from blood were motile, but culture in vitro for 1-3 days increased the proportion of locomotor forms to 30-40%. These cells were placed on 3-D collagen gels, and a chemotactic source was presented nearby on a small filter placed on the surface of, or within, the gel. The minority of lymphocytes that were capable of locomotion showed chemotactic responses to filters soaked in lipopolysaccharide if fresh human serum (20%), but not heat-inactivated serum, was present. Lymphocytes responded by protrusion of a lamella in the direction of the gradient source: 76% of locomotor lymphocytes showed their first orientation into the 180 degrees sector facing the source. They then moved directionally towards the source. The response to purified C5 peptides was equivocal. The locomotor lymphocytes showed a chemotactic response to supernatant fluids derived from cultures of the adherent mononuclear cell fraction from human blood (greater than 80% monocytes), judged by the same criteria. No particular lymphocyte type constituted the locomotor population. After exposure to LPS-activated serum, both T and B lymphocytes showed locomotor forms. There were slightly more T4+ cells among the locomotor population than among the population as a whole.

Assays of leukocyte locomotion and chemotaxis.

This review discusses the range of methods which are currently available for measuring locomotion and chemotaxis of leukocytes in vitro, their history, and some definitions of terms. Assays of the net migration of large cell populations, such as the filter assay are the most popular and are useful for identifying chemoattractant molecules, but give no direct information about how these molecules influence the speed and direction of cell movement (chemokinesis and chemotaxis). Visual assays including measures of orientation in gradients and time-lapse filming give detailed information about cell paths and direct evidence for chemotaxis and chemokinesis. The polarization assay is a useful visual screening assay. Assays which simulate the situation in living tissues are becoming more popular and include migration through collagen or fibrin gels or through monolayers of vascular endothelium. Locomotion is a complex process, no single assay gives full information and the use of more than one assay is recommended.

Evaluation of methods for isolating human peripheral blood monocytes. Studies on chemotactic locomotion and other functional characteristics.

We have compared six different methods of purifying human blood monocytes for their usefulness in relation to assays of polarization, locomotion and chemotaxis. For polarization assays it is essential to prepare an unstimulated, spherical, cell population in suspension. The techniques compared were based either on density differences between monocytes and lymphocytes using Percoll or Nycodenz, or on the separation of adherent monocytes from non-adherent cells on protein-coated surfaces, i.e., foetal calf serum (FCS); gelatin-FCS; gelatin-plasma; baby hamster kidney (BHK) microexudate coats. The BHK microexudate technique (Ackerman and Douglas, 1978) gave the best yield and purity of monocytes. These were spherical and had not been activated by the separation procedure. This technique provided monocytes in suspension that were functionally normal in locomotion and chemotaxis assays, phagocytosis, chemiluminescence and Fc receptor expression. To achieve a good yield of spherical cells, it was necessary to use tubes to which monocytes did not adhere. Siliconized glass was superior to tissue culture plastic for this purpose.

Methods for phenotyping polarized and locomotor human lymphocytes.

Lymphocytes show heterogeneity both in phenotype and in locomotor activity; methods which permit the simultaneous assessment of both are therefore useful. The activation of locomotion may be dependent on interactions between lymphocytes and accessory cells, making it necessary to study mixed cell populations. We describe here two in vitro procedures which do this. Firstly the lymphocyte polarization assay has been adapted by using the alkaline phosphatase-anti-alkaline phosphatase method (APAAP) after fixation with glutaraldehyde. Secondly, we have allowed lymphocytes to invade collagen gels and then digested the gel with collagenase to recover the locomotor population. This can then be phenotyped by immunofluorescence using a fluorescence-activated cell sorter (FACS). Collagenase did not affect the staining pattern with commonly-used markers such as CD3, -4, -8, -19, -29, -45RO and -45RA.

The use of collagen or fibrin gels for the assay of human neutrophil chemotaxis.

Neutrophil leucocytes are known to migrate actively into 3-dimensional gels of collagen or fibrin. In this paper, we have used such gels to study chemotaxis of human blood neutrophils towards gradient sources of formyl-methionyl-leucyl-phenylalanine (FMLP) using 2 assay systems. The first resembled the micropore filter assay in that neutrophils on the upper surface of collagen gels were allowed to invade in the presence of either an isotropic concentration or a gradient of FMLP. Neutrophils invaded the gel vigorously in both cases. The effect of the gradient was assessed by determining the population distribution at different levels in the gel. Cells moving randomly should be distributed normally, and directional locomotion should cause deviation from normal distribution. Such a deviation was seen, but was of marginal significance. A more direct demonstration of chemotaxis was achieved by the second assay in which an agarose slab containing FMLP was incorporated into a gel, and the paths of nearby neutrophils were filmed. These cells showed an unequivocal directional response to the FMLP gradient. Protein gels can thus be used in the same way as both the presently used filter assays and visual assays using plane substrata, but with the advantage of providing a more physiological environment for the study of chemotaxis than either.

Separation of mouse lymphoblasts by discontinuous density centrifugation on Percoll gradients.

Mouse lymphoblasts generated in vivo by a topical application of the contact sensitizer oxazolone or by the contents of the gut lumen were separated by discontinuous density centrifugation on Percoll gradients. A 3-step gradient was used to divide the cells into two subpopulations. For cells from oxazolone stimulated lymph nodes, the low density band contained 20-30% of the initial cell number applied to the gradient; 25-40% of this population were in S phase and nearly all the large and pyroninophilic cells were confined to this layer. The high density step cells (70-80% of initial cell number) were predominantly small lymphocytes with less than 0.5% in S phase. Similar results were obtained using cells from picryl chloride stimulated lymph nodes or from mesenteric lymph nodes.

Visual observations of chemotaxis and chemotropism in mouse macrophages.

Locomotion of mouse macrophages in relation to spores of Candida albicans in mouse serum was studied by time-lapse cinematography using thioglycollate-elicited peritoneal macrophages, resident peritoneal macrophages, and a murine macrophage line (J774 B10). Thioglycollate-elicited macrophages and the cell line responded well to the spores, but very few of the resident macrophages showed any response. The macrophages showed chemotactic responses with straight-line locomotion towards spores. Thioglycollate-elicited macrophages, which were frequently spread on glass, could also respond to spores 20-30 micrometers away by chemotropism, i.e. extension of a large hyaline veil to engulf the spore without prior displacement of the body of the cell. Engulfed spores were then pulled into the organelle-rich cell centre. In this population, there was no incompatibility between spread morphology and motile behaviour. In contrast, J774 B10 did not spread and moved with a rounded morphology and with a very small anterior hyaline veil towards the gradient source. Macrophages moved more slowly than peripheral blood neutrophils and monocytes. The mean speed of thioglycollate-elicited and cell line macrophages was 3-4 micrometers per minute. The few motile resident peritoneal macrophages moved even more slowly, i.e. ca 2 micrometers per minute.

Locomotion and chemotaxis of lymphocytes.

The behaviour of locomotor T and B lymphocytes and the chemoattractants to which they respond in vitro are reviewed. Following activation, T cells respond by locomotion and chemotaxis to cytokine attractants including IL-15 and IL-2 and several chemokines. In activated B cells chemotaxis may be signalled through the antigen receptor. Conversely resting lymphocytes respond poorly to the above signals though their locomotion is activated by contact with high endothelial venular cells. These differences in locomotion between resting and activated lymphocytes, together with differences in adhesion, may explain why activated lymphocytes migrate preferentially into inflammatory sites while resting cells recirculate.

Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes.

The effects of staphylococcal products as chemo-attractants for human blood neutrophils and monocytes and as inhibitors of locomotion of these cells were studied with bacterial cells, culture filtrates and isoelectrically focused fractions from culture filtrates of nine strains of Staphylococcus aureus. Little direct chemotactic activity of staphylococcal products for neutrophils was observed, although a chloroform-soluble extract of the whole organisms contained such activity. The major chemotactic effect of staphylococci for neutrophils was indirect, i.e., generated when the organisms or their products were incubated with plasma, perhaps due to activation of complement. In contrast, direct chemotactic activity for monocytes was found in a large number of staphylococcal fractions. Staphylococci also produced inhibitors of locomotion of both neutrophils and monocytes. Isoelectric focusing showed more fractions inhibitory for neutrophils than for monocytes. Some of the inhibitors could be identified. Staphylococcal alpha-toxin inhibited migration of both neutrophils and monocytes. Sphingomyelinase C (beta toxin) inhibited migration of monocytes but not of neutrophils. Leucocidin-rich strains were strongly active as inhibitors of neutrophil locomotion but less so as inhibitors of monocyte locomotion.

Effects of anaesthetics on membrane mobility and locomotor responses of human neutrophils.

The morphological response of neutrophils to chemotactic factors is characterized by an immediate change (in seconds) from a spherical to an irregular shape. Within two or three minutes, the cells assume the head-tail polarity typical of locomotor cells. In this study the effects of the anaesthetic drugs, propofol and thiopentone, on the time-sequence of the morphological response of human neutrophils to the chemotactic peptide fMet-Leu-Phe were examined. At concentrations seen in the plasma during anaesthesia, both drugs inhibited both the rate and degree of the neutrophil chemotactic response. The effect of propofol was not attributable to its lipid vehicle, as 10% intralipid alone had no effect on neutrophil polarization. Plasma membrane reorganization occurs during polarization of neutrophils, resulting in morphological and functional changes which prepare the cells for chemotaxis and phagocytosis. Fluorescence recovery after photobleaching (FRAP) was used to investigate effects of the anaesthetics on membrane lipid behaviour. With a lipid probe, the proportion of mobile lipid in neutrophils exposed to propofol or thiopentone was reduced. There was a less significant reduction with intralipid which also caused reduction in velocity of lateral diffusion of the probe. These findings suggest that the inhibitory effects of anaesthetics on neutrophil locomotion are related to reductions in fluid mobility of the plasma membranes of anaesthetic-treated cells.

Cell-membrane activation of macrophage function.

A number of short-term macrophage functions are stimulated immediately on contact of suitable external materials with the macrophage plasma membrane. These include locomotion and chemotaxis, phagocytosis, and exocytosis of lysosomal hydrolases. They are probably contractile events and may be initiated by a transient increase in permeability to divalent cations either of the plasma membrane or of hypothetical intracellular cations stores. Such permeability changes may follow interaction of hydrophobic substances with the membrane bilayer or follow contact with substances which induce clustering of membrane proteins. It is conjectured that similar substances might induce long-term events such as DNA and protein synthesis, mitosis and activation of cytotoxic function in macrophages, but that to do this, contact of the substance with the macrophage must be prolonged.

Lymphocyte locomotion "in vitro": the role of growth activators and chemoattractants.

Studies of lymphocyte locomotion in vitro are reviewed. This locomotion is important (a) for recirculation and the traversing of high endothelial venules in lymphoid tissue; (b) for recruitment of lymphocytes into inflammatory sites. In the latter situation, activated lymphocytes migrate more actively than resting lymphocytes. Our studies indicate that lymphocyte activators such as PHA, anti-CD3 antibodies, or the Cowan staphylococcus confer locomotor capacity on populations of human blood lymphocytes which, in the resting state, are immotile. Locomotor capacity is acquired in the G1 phase of growth and requires protein and RNA synthesis but not DNA synthesis. Anti-CD3-driven locomotor activation is inhibited by cyclosporin A, suggesting that new gene expression is required. The mitogens do not act directly as locomotor stimulants, i.e. they are not themselves chemotactic or chemokinetic factors. Rather they activate the potential for motility of lymphocytes and also cause release of lymphokines which are the direct stimulants for locomotion. One of these lymphokines (lymphocyte chemotactic factor: LCF) has been partially characterized.

Locomotion and chemotaxis of mononuclear phagocytes.

The capacity for locomotion and for chemotaxis is probably very different in monocytes and macrophages from different sources. Numerous techniques have been established for studying the locomotion of these cells. Many of the factors are sparsely documented and the reports are scattered among various cell types. Heterogeneity of locomotion and chemotactic responsiveness is evident when established macrophage lines and mouse peritoneal macrophage are studied. The effects of mononuclear phagocytes and their released products on the locomotion of other cell types are reviewed.

Effects of fluoride on locomotion of human blood leucocytes in vitro.

The effect of NaF on the locomotion and chemotaxis of human blood neutrophils and monocytes was studied using two assays: the micropore filter assay and a time-lapse cinematographic assay in which the chemotaxis of cells in response to spores of Candida albicans was filmed. At high concentrations (greater than 10(-4) M), NaF inhibited locomotion of both cell types, but no inhibition of locomotion of either cell-type was seen in either assay using NaF at less than or equal to 10(-4) M, whether or not the cells were responding to a chemotactic source. This was so, even for monocytes incubated for 48 h in the presence of NaF. It is therefore improbable that fluoride, at levels added to drinking water or found in the body fluids of persons drinking fluoridated water, has any deleterious effect on the locomotor capacity of phagocytic cells or on their capacity to detect and home on to chemotactic sources.

Serum IgG and IgE antibody against aerosolised antigens from Nephrops norvegicus among seafood process workers.

Employees in a seafood factory developed high titers of serum IgE and IgG antibody to antigens from prawn (N. norvegicus) which were aerosolized during processing. Significant serum IgE antibody titers occurred only among those subjects with occupation-related respiratory symptoms, and this serological parameter may be a useful clinical adjunct in the investigation of this disease. Serum IgG antibody was detected with equal frequency and titer in symptomatic and asymptomatic workers. There was no significant correlation between either antibody class response and the individual's age or years of work exposure. Cigarette smoking, however, was positively associated with the IgE antibody response, and negatively associated with the IgG antibody response to the same inhaled antigen. Investigating the effects of smoking at the mucosal level in the lung may provide insight into how the lung processes and responds to inhaled antigens.