Phorbol myristate acetate induction of chemotactic migration of human polymorphonuclear neutrophils.

The in vitro migration of human polymorphonuclear neutrophils (PMNs) was studied employing an enzymatic assay of cell migration with phorbol myristate acetate (PMA) as the test stimulant. Our data clearly show that PMA in concentrations between 1 and 100 ng/ml in the lower wells of blind-well chambers induced chemotactic migration. Chemokinesis (increased migration) was not induced when PMA was present in both the upper and lower chambers (i.e., in a nongradient mode). Clearly our data indicate that PMA is chemotactic for human PMNs and, coupled with published studies of the effect of PMA on PMNs, suggest activation of an intracellular gradient of membrane-associated protein kinase C as a possible new mechanism for the induction of oriented migration of PMNs. Such a mechanism may be generalized to include membrane-soluble materials (e.g., inflammatory mediators, microbial products), which establish internal gradients of activated PKC rather than via the "classic" agonist-surface receptor mechanism, providing an alternative pathway for the induction of leukocyte chemotaxis.

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin.

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.

An enzyme-assessed microplate-assay for neutrophil adherence. I. IgA-induced adherence of human PMNs.

The binding of PMNs to extracellular matrix and cells is crucial to PMN host defense. Adherence mechanisms and the many families of molecules involved are major areas of study. We present here details of an enzyme-assessed microtiter plate assay for neutrophil adherence. This assay uses low numbers of cells (50,000/well) and permits analysis of several hundred wells in a short period of time, by using an ELISA reader. With this assay we observed 5- to 10-fold increases in the number of adherent human PMNs in response to nanogram amounts of LPS or as little as 5.0 micrograms/ml of aggregated IgA. Although fluoride blocked the LPS-induced adherence response, IgA-induced cell binding was largely unaffected.

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Fluoride activation of neutrophils: similarities to formylmethionyl-leucyl-phenylalanine.

Fluoride induced degranulation of both primary and specific granules from neutrophils pretreated with cytochalasin B. There was a similarity in the dependency on extracellular Ca2+ for fluoride- and for FMLP-stimulated O2- generation and degranulation. Pertussis toxin, but not cholera toxin, inhibited FMLP and fluoride activation of neutrophils, while neither toxin affected PMA activation of these cells. These results suggest that fluoride and FMLP activate neutrophils through a common Ca2+-dependent and pertussis toxin-sensitive pathway.

Aggregated human colostral sIgA stimulates delayed, non-complement-dependent, NBT reduction by human neutrophils.

Antibodies often alert polymorphonuclear neutrophils (PMNs) to the presence of pathogens. In a study to learn if secretory immunoglobulins can carry out this function, we observed that as little as 4 micrograms/ml of secreted human immunoglobulin A from colostrum (sIgA), in the absence of antigen, stimulated human PMNs to reduce nitroblue tetrazolium (NBT). NBT reduction was inhibited 71% by superoxide dismutase. Active complement pathways were not required since comparable activity was obtained in the presence of heat-inactivated serum. Aggregated forms of sIgA were much more stimulatory than nonaggregated dimeric sIgA. Such interaction between PMNs and sIgA could act in situ to enhance protection against infections of exposed body sites or could initiate inflammatory tissue damage.

Role of extracellular calcium in neutrophil responsiveness to chemotactic tripeptides.

Cellular stores of Ca2+, but not extracellular Ca2+, are required for effective FMLP stimulation of neutrophil O2- production and degranulation. Neutrophils transferred from Ca2+-containing to Ca2+-free medium gradually lose their responsiveness to FMLP, such that after 40 min in the Ca2+-free environment they have lost 60-70% of their initial responsiveness to FMLP. The loss in responsiveness is reflected both in an increase in lag interval and decrease in velocity of O2-synthesis. The rate of decline in responsiveness to FMLP is greatly accelerated when neutrophils incubated in the presence of A23187 and Ca2+-free medium, while the rate of loss of responsiveness to FMLP is not affected by EGTA but the extent of loss is increased. Gradual recovery of FMLP-induced O2- generation occurs when cells are transferred from Ca2+-free to Ca2+-containing medium. PMA-induced neutrophil O2- generation is not influenced by the presence or absence of extracellular Ca2+. It is our view that the rise or fall of neutrophil responsiveness reflects repletion or depletion of cellular stores of Ca2+ essential for stimulus-effector coupling and that the role of extracellular Ca2+ is subservient to maintenance of these stores.

Modulation of the kinetics of induced neutrophil superoxide generation by fluoride.

Fluoride (F-) is unique in that, depending upon concentration, it can either stimulate or inhibit the synthesis of the neutrophil's major intracellular microbicidal product, superoxide anion (O2-). While a number of studies have delved into F- induction of O2- generation, little has been reported on the effect of F- on the synthesis of O2- induced by other agents. In this report, we show that F- inhibits the activation and activity of neutrophils induced either by formyl-methionyl-leucylphenylalanine (FMLP) or by phorbol myristate acetate (PMA). The extent of increase in lag period and decrease in velocity of O2- synthesis was found to be directly related to the concentrations of F- and of hydrogen ion in the medium and to the length of contact time between the neutrophil and F-. Decreasing the pH of the medium, while maintaining a constant concentration of F-, disproportionately increased the inhibitory effect of the anion over the effect of pH alone. Fluoride inhibited the responsiveness of neutrophils to FMLP more than it did their responsiveness to PMA. A gradual but partial reversal of F-'s inhibition of O2- synthesis could be achieved by transferring inhibited cells to F(-)-free medium. Our study shows that the suppression of both FMLP- and PMA-induced synthesis of O2- by neutrophils is most dramatic at a reduced pH and is probably related to the intracellular accumulation of F-.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses.

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.

Activated rat neutrophils. A sequential quantitative assay for aggregation and NBT reduction.

We present here a rapid, sensitive, and convenient approach for the analysis of activated Lewis rat PMNs based on detecting separately, or in tandem, PMN aggregation and PMN reduction of nitroblue tetrazolium (NBT). These responses are quantitated using an ELISA scanner which can rapidly measure optical densities of cell cultures in microtiter plates. Aggregation induced by as little as 0.005 micrograms/ml of phorbol myristate acetate (PMA), 0.01 micrograms/ml lipopolysaccharide (LPS), or a 1:160 dilution of lymphokine-containing rat serum can be detected employing this approach. NBT reduction was induced by as little as 0.01 micrograms/ml PMA. Blocking studies employing 2-deoxyglucose, iodoacetamide, and polymyxin B gave the expected results and confirmed that these assays detect cellular responses to soluble stimuli. Using this technology the effects of PMA and LPS on rat peritoneal exudate PMNs were evaluated. Rat PMNs appeared less sensitive to LPS than human PMNs and also reduced NBT more slowly following stimulation with PMA. Because of the slowness in NBT reduction following stimulation, NBT reduction can be evaluated, in tandem, after measuring aggregation. The simplicity of this system, coupled with the speed with which large numbers of microcultures can be read and the low number of cells required, make this approach for studying responses especially attractive.

Ontogeny of T-cell mitogen response in Lewis rats: II. Early appearance and loss of suppressor activity.

Spleen cells from rats 2 to 132 days old were cultured with 1-125 micrograms/ml Concanavalin A (Con A). At high doses of Con A, the high spontaneous thymidine uptake of spleen cells from rats 15 to 21 days old was suppressed, whereas spleen cells from younger rats showed no suppression of spontaneous mitogenesis at equally high Con A doses. Removal of either plastic-, nylon wool-, or carbonyl iron (cFe) adherent cells not only removed suppression of background by high Con A doses, but also allowed mitogenic responses at adult levels in normally unresponsive 15 to 21 day old pups. Low doses of X-irradiation did not cause a similar loss of suppression. We suggest that although there is an influx of ConA responsive cells into the rat spleens at 15 to 16 days, the mitogen responses of these cells are suppressed by an adherent cell population which is activated by high doses of Con A.

Ontogeny of T-cell mitogen response in Lewis rats: I. Culture conditions and developmental patterns.

Spleen and thymus cells from female Lewis rats 2 to 220 days old were cultured with phytohemagglutinin (PHA) or Concanavalin A (Con A). The optimal dose of Con A for mitogenesis ranged from 1 microgram/ml for suckling rats to 125 micrograms/ml for adult rats. Thymus cells differed from spleen cells in that they required 72 hours for maximum thymidine incorporation; whereas spleen cells attained maximum incorporation within 48 hours. Thymidine uptake in unstimulated spleen cell cultures was high in the neonate and decreased with age. Maximum splenic cell responses to Con A increased from very low levels in the 2-day old to adult levels by 3 weeks coincident with weaning. Maximum PHA responses of splenocytes matured more slowly, reaching adult levels by 8 weeks. Thymus cell responses to Con A and PHA were mature from birth.

Endogenous component chemotactic assay (ECCA).

We have developed a chemotactic assay in which migrated cells are quantitated by measuring levels of an endogenous cellular component. The endogenous component chemotactic assay (ECCA) employs standard double-membrane, blind-well methodologies but is unique in that leukocyte migration is quantitated by measuring lactic dehydrogenase (LDH) activity endogenous to cells that have migrated. This approach avoids the tedium of microscopic counting as well as the problems associated with cell-labeling techniques. Using the ECCA technique we have shown: (1) that N-formylmethionyl-leucyl-phenylalanine (fMLP) is both chemokinetic and chemotactic for human polymorphonuclear neutrophils (PMNs); (2) that both incubation time and starting PMN density affect the proportion of cells that migrate; (3) that approximately 30% of the available PMNs eventually migrate; and (4) that PMN "fall off" from membranes, readily detectable by this assay, is affected by starting PMN density, incubation period, and nature of the attractant. The technique as presented can detect migration when a starting cell density as low as 7 x 10(4) PMNs/well is employed and can be made more sensitive by increasing the period over which LDH is allowed to act. Considerable potential exists to further apply the ECCA concept to the study of the migration of subpopulations of cells in mixtures by assaying for distinguishing endogenous cellular markers.

A rapid quantitative assay for activated neutrophils.

We present here a rapid, sensitive, and convenient assay for activated human PMNs based on detecting the decreased optical density (OD) of aggregated cell suspensions. This quantitative assay uses an ELIZA machine to measure OD changes, with time, of activated cells (5 X 10(5) cells/well) in microtiter plates. The assay is sensitive, detecting aggregation induced by as little as 0.0001 microgram/ml of LPS, or lymphokines in Con-A-activated supernatant diluted 1/500. The assay permits analysis of 400 separate PMN suspensions on the same day starting with less than 80 ml of blood.

Induction of T-lymphocyte responses to a small molecular weight antigen. III. T-T cell interactions to determinants linked together: suppression vs. enhancement.

The experiments presented in this paper demonstrate the existence of T-T cell interactions in responses to azobenzenearsonate (ABA)-protein conjugates, and also make the point that the spectrum of T-cell regulation from facilitation (i.e., help) at one end to suppression at the other, which has been well documented in T-B cell interactions, is also followed in T-cell regulation of other T lymphocytes. The data extend the activity of ABA-specific suppressor cells, which were shown to specifically suppress the development of delayed hypersensitivity to ABA-T, to T cells responsible for delayed hypersensitivity to protein antigens provided immunization is carried out with ABA conjugates of these antigens. Thus, suppressor T cells acting on the development of delayed hypersensitivity are not limited in their effects to T cells bearing the same specificity but can effectively suppress responses on immunologically unrelated T cells if they are specific for carrier antigens covalently linked to the ABA-T determinant. Moreover, these studies demonstrate that, as is true of T-B cell interactions, the most efficient T-T cell interactions occur to determinants linked together on the same molecule thus supporting the concept that development of effector T-cell function may involve participation of at least two distinct precursor cells, each of which may convey independent determinant specificities and/or genetic control.