Initiating Cell Activity Research Articles

Nonsteroidal antiallergic treatments in allergic rhinitis.

Allergic rhinitis is characterized by an inflammatory reaction consequent to an initial IgE-dependent mast cell activation. The conventional treatment is mainly based on antihistamine and topical corticosteroid prescriptions. In this article the authors consider the antiallergic-antiinflammatory activity exerted by some nonsteroidal drugs or immunologic treatments. Clinical and experimental evidence demonstrates that these treatments are able to control allergic inflammation in patients with allergic rhinitis. In conclusion, treatment of allergic rhinitis may also be based on the prescription of nonsteroidal drugs with antiallergic activity.

CD28 Costimulation Accelerates IL-4 Receptor Sensitivity and IL-4-Mediated Th2 Differentiation

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.

Expression of interferon-γ by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity

Several lines of evidence suggest that overexpression of interferon γ (IFN-γ) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-γ by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-γ. The effect of IFN-γ on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-γ vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-γ–expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-γ or control vector. Marrow cells transduced with either vector were transplanted into W/W v recipient mice. The percentage of vector-containing cells in the mIFN-γ mice was significantly lower than in the control mice, suggesting that mIFN-γ–transduced primitive cells may not have survived culture, or that mIFN-γ directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-γ–transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-γ group. These data indicate that ectopic or overexpression of mIFN-γ, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo. Published by Elsevier Science Inc.

New Horizons in Orthopaedic Research

The report by Nakashima et al.6 in this issue of The Journal and the one by Blaine et al.3 in the October 1997 issue dealt with signal transduction mechanisms in macrophages stimulated by orthopaedic biomaterials. Although these pioneering studies represent initial findings and focus on only a narrow range of possible signaling events, they open new vistas within clinically oriented orthopaedic research. The ultimate aim of this new line of research is to delineate the fundamental cellular mechanisms of periprosthetic osteolysis induced by particulate wear debris and to establish strategies for the development of therapeutic agents to prevent, reverse, or retard this adverse outcome of joint replacement procedures. Furthermore, the study of cellular signal transduction pathways can be applied to numerous areas of orthopaedic research, including the growth and development of cartilage and bone, the pathogenesis of osteoarthritis, fracture-healing, and tumor growth, to name but a few. One of the most important advances in biomedical science in this decade is the identification of intracellular signaling events as part of cellular adaptive responses. These regulatory events are important for mammalian cells to deal with changes in their microenvironment and maintain homeostasis. Cells receive external signals by way of surface membrane receptors, and as a consequence certain amino acids (usually tyrosine, serine, or threonine residues) of the cytoplasmic tail of the receptor become phosphorylated. This initial biochemical event is amplified through a series of protein kinase phosphorylations that in the end trigger a defined set of intracellular adaptive processes, including long-term gene responses. Extracellular compounds in the form of lipids, hormones, growth factors, cytokines, chemokines, counter receptors of other cells, or other cellular mediators can bind to specific cell-surface receptors. These interactions trigger intracellular signal-transduction cascades that result in the modulation of nuclear transcription factor activity. Transcription factors are nuclear …

Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection.

Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-gamma from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-alpha production is induced by day 3 post infection. Not only do these data satisfactorily explain the differences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.

Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection

Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-γ from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-α production is induced by day 3 post infection. Not only do these data satisfactorily explain the dfferences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.

Short term fasting does not aggravate immunosuppression in harbour seals ( Phoca vitulina) with high body burdens of organochlorines

Two groups of 11 harbour seals ( Phoca vitulina) with different body burdens of organochlorines were subjected to an experimental 15-day fasting period, during which they lost an average 16.5% of their body weights. Blood levels of the most persistent organochlorines showed an approximate twofold increase, while levels of aryl hydrocarbon receptor-binding organochlorines remained largely unaffected. Few differences in immunological parameters were observed between the two dietary groups. Numbers of circulating lymphocytes dropped to about 65% of the initial values and NK cell activity showed a slight increase in both groups. Mitogen- and antigen-induced lymphoproliferative responses of the Baltic group of seals remained within normal ranges. These results suggest that relatively short-term fasting periods do not present an additional immunotoxicological risk to seals with high body burdens of organochlorines.

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.

In humans and experimental animals the presence of bacterial lipopolysaccharide (endotoxin, LPS) signals the presence of gram-negative bacteria. Recognition of LPS triggers gene induction by myeloid and nonmyeloid lineage cells. These inducible genes encode proteins that include cytokines, adhesive proteins, and enzymes that produce low molecular weight proinflammatory mediators. Together the products of these inducible genes upregulate host defense systems that participate in eliminating the bacterial infection. Unfortunately, these same mediators contribute to a serious human disease known as septic shock. Considerable progress has been made during the past decade in determining the sources, identities, and sequence of release of these mediators. In contrast, until recently, marked gaps in our knowledge existed regarding the identity of the LPS receptor and intracellular signaling pathways responsible for LPS-induced cell activation. The discovery in 1986 of a plasma protein termed LPS binding protein (LBP) led to the discovery of unanticipated mechanisms of LPS-induced cell activation. CD14 was found as a soluble serum protein or as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells; it now occupies a key role in LPS-induced cell activation as we understand it today. Here we discuss how LBP enables LPS binding to CD14 and how complexes of LPS and soluble or GPI-anchored CD14 participate in cell activation. We also review the evidence supporting a model for a functional LPS receptor of myeloid cells, which is multimeric, comprised of GPI-anchored CD14 and a presently unidentified transmembrane protein that together bind LPS and initiate cell activation via kinase cascades.

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

THE THROMBIN RECEPTOR

1. The thrombin receptor has now been cloned and found to be a member of the G-protein-coupled seven-transmembrane domain receptor family. 2. The receptor has been detected directly in platelets, endothelial cells and smooth muscle cells and studies using receptor-derived peptides have demonstrated that this receptor may be the one responsible for many of the actions of thrombin in platelets, endothelial cells, smooth muscle cells, fibroblasts, mesangial cells and neural cells. 3. The receptor appears to be activated by the novel mechanism of cleavage by thrombin to yield a new N-terminus which then interacts with the receptor as a tethered ligand to initiate cell activation.

Mechanism of lipopolysaccharide-mediated transcriptional enhancement of the mu gene.

LPS induces both B cell proliferation and differentiation to Ig secretion. By treating stimulated cells for a brief period with staurosporine, and inhibitor of protein kinase C, it is possible to allow continued proliferation but partially inhibit differentiation. Analysis of the molecular basis for the decrease in IgM production shows that the increased transcription of the Ig-H chain gene induced by LPS is abrogated by staurosporine treatment whereas alteration of 3' end processing is not affected. These experiments indicate that LPS continues to mediate its effect on some of the more distal differentiative events through protein kinase C even after initial cell activation.

Heterogeneity of natural killer cell subsets in NK-1.1 + and NK-1.1 − inbred mouse strains and their progeny

The 4LO3311 monoclonal antibody, a new NK-specinc reagent recently produced in our laboratory, reacts with spleen cells of 11 mouse strains, most of which do not express the NK-1.1 alloantigen recognized by the PK136 mAb. Among positive strains, the susceptibility of spleen cells to the complement-dependent NK-inhibiting activity of the 4LO3311 mAb was variable but independent of the initial NK cell activity level of cells tested. This property was furthermore not modified after poly(I:C) stimulation. The susceptibility of spleen cells to the in vitro 4LO3311 mAb plus complement treatment is however influenced by the absolute number of 4LO3311 + cells as well as by the density of the corresponding alloantigen at the cell surface. Moreover, it was established that the strain-related variations observed also depended upon the relative size of the 4LO3311 cell subset within the lytic NK cell population. Indeed, when C3H (NK-1.1 −4LO3311 +) mice were inoculated with the 4LO3311 mAb, the lytic activity of their spleen cells was almost unaltered but 4LO3311-reactive cells were no longer detected in the spleen of treated animals and remaining NK cells were totally resistant to the in vitro 4LO3311 mAb plus complement treatment. These findings indicate that the 4LO3311 mAb identifies a subset rather than all NK cells, even in a NK-1.1 − strain. Since a NK-1.1-unreactive cell subset was identified in NZB (NK-1.1 +4LO3311 −) mice inoculated with the PK136 mAb, the NK-1.1 + cell population is not necessarily responsible for all the splenic NK cell activity in all NK-1.1 + strains. In B6C3F 1 hybrid mice, a relatively large subset of NK-1.1 −4LO3311 − cells was found in addition to those expressing the NK-1.1, the 4LO3311 alloantigen, or both. According to these results, NK cell heterogeneity should thus be taken as an evolving concept whose resolution appears more and more complex with the identification of new NK-specific reagents.

Muramyl dipeptide-induced changes in murine splenocyte responses to concanavalin A

The effect of muramyl dipeptide (MDP) on Con A-stimulated activation of murine spleen cells was studied. MDP was found to enhance or suppress the proliferative response of splenocytes when different concentrations of Con A were used. MDP was shown to change the IL-2 content in culture supernatants of stimulated cells and to influence IL-2-dependent proliferation of Con A-blasts. A high degree of correlation was found between the proliferation of Con A-blasts and the expression of IL-2 receptors on Con A-blasts. This correlation, however, disappeared in the presence of MDP. The effects of MDP were shown to depend on the level of initial cell activity or rather on conditions leading to a given initial activity of cells.

PHOTOSENSITIVE PHENOMENA OF NITRILE HYDRATASE OF Rhodococcus sp. N‐771

Abstract— When the washed cells of Rhodococcus sp. N‐771 were incubated at 5°C in the dark under aerobic condition, their nitrile hydratase was inactivated after several days. Most of this activity was recovered by light irradiation. The speed of inactivation in the dark was affected by incubation temperature and amount of oxygen supply. Under anerobic conditions, however, this reversible dark inactivation was not observed; the photoirradiation of the cells irreversibly inactivated the initial cell activity by about 15%. The enzyme activity of the cell‐free extract of the inactivated cells can also be recovered when photoirradiated. This process did not require oxygen, and was not prevented by dialysis. However, the enzyme of the cell‐free extract could not be inactivated by dark, aerobic incubation nor by photoirradiation after dark anaerobic incubation.

Signal transduction in cells following binding of chemoattractants to membrane receptors.

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.

Enrichment of murine splenic natural killer (NK) cells by the sequential elimination of non-NK cells

A simple and reliable three-step procedure to enrich for murine endogenous splenic NK cells is described. The method is based on the sequential elimination of non-NK cell subsets by standard and inexpensive techniques executed in a specific order. First, macrophages and other adherent cells are eliminated by incubation on plastic surface. Secondly, the T cells are excluded from the multicellular aggregates formed by agglutination of the remaining cells with wheat germ lectin. Thirdly, after dissociation of the aggregates with N- acetyl- D-glucosamine and osmotic lysis of erythrocytes, NK cells are separated from other nucleated cells by nylon wool filtration. C57BL/6 spleen cells were used to establish the enrichment procedure. Usually their NK cell activity is intermediate but occasionally either low or high NK cell activity was observed in input cell suspensions. The NK cell activity recovery and the degree of enrichment varied inversely with the initial NK cell activity level of the input cell suspension. When initial NK cell activity was intermediate, it was enriched 10–30-fold. Experiments were done to establish if suppressor cells, and nylon wool-adherent, naturally activated NK cells, putatively present in input cells, could have been responsible for the abnormal initial NK cell activity detected in some C57BL/6 spleen cell suspensions and for the variations in the degree of enrichment achieved by the method here described. Either no or negligeable suppressor cell activity was noted in the cell fractions normally discarded at each step of the procedure. On the other hand, nylon wool-adherent NK cells were eliminated during the fractionation of spleen cells with higher than average initial NK cell activity and would account for the lower NK cell enrichment obtained in these conditions.

Extracellular but not intracellular application of peptide hormones activates pancreatic acinar cells

Peptide hormones, like neurotransmitters, are traditionally thought to activate cells by interacting with receptor sites accessible only from the extracellular space. However, there is no available evidence that establishes whether intracellular injections of peptide secretagogues can or cannot initiate cell activation. In view of recent demonstration that peptide hormones can penetrate the intracellular space in some tissues and the reports that intracellular injections of the neurotransmitter, dopamine, into acinar cells of cockroach salivary gland cause stimulation it seems of fundamental importance to test directly whether introduction of peptide secretagogues inside acinar cells of mammalian exocrine tissue can induce cell activation without first interacting with the outer surface of the external cell membrane. The data presented here show that injections of the secretagogue peptides caerulein and bombesinnonapeptide (bombesin-NP) into pancreatic acinar cells fail to evoke the characteristic potential and conductance changes that are observed following extracellular applications of these peptides.

The effect of nutritional state on the lipoprotein lipase activity of isolated fat cells

The lipoprotein lipase activity of rat epididymal adipose tissue falls on starvation and increases on refeeding. Studies with fat cells isolated from this tissue have shown that increases in the activity of the enzyme occur under appropriate incubation conditions in vitro. The present study compares the responses of cells isolated from the adipose tissue of fed and 48-h starved rats. Fat cells from rats starved for 48 h display a lower initial lipoprotein lipase activity than cells from fed rats. When cells from rats in both nutritional states are incubated in a suitable medium at 25° C, there is a progressive increase in the medium lipoprotein lipase activity. The absolute increase in the total activity of the incubation system during incubations of cells from 48-h starved rats is significantly less than during incubations of cells from fed rats. However, when expressed as a percentage of the initial cell activity, the rises in total activity are similar in the two nutritional states. Cycloheximide has no significant effect on the increase in activity of lipoprotein lipase that occurs with cells from 48-h starved rats. However, it does partially block the increase in activity seen with cells from fed rats and in a manner similar to that previously reported for cells from 24-h starved rats. The significance of the results is discussed in relation to previous studies with both intact adipose tissue and isolated fat cells.

The Activation of Lymphocytes by the Ionophore A 23 187

Abstract The possible mechanism by which a divalent cation ionophore, A 23 187, initiated the activation of lymphocytes was investigated. Rabbit lymphocytes were cultured in the presence of the ionophore, the T cell mitogen Con A, or the B cell mitogen GARIG serum. The proliferative response, assessed by incorporation of 3H-thymidine into DNS, as well as the increase in incorporation of 3H-uridine induced by the ionophore was comparable to that caused by the receptor-binding B mitogen in T-B mixtures. Thymocytes responded weakly and inconsistently to the ionophore, whereas purified B lymphocytes proved to be highly reactive, suggesting that A 23 187 is primarily a B mitogen in rabbits. Removal of macrophages did not impair the activating capacity of the ionophore. To initiate cell activation the ionophore had to be present for at least some hours. At optimal concentrations of Ca++ (1 mM), increasing doses of Mg++, which has the same binding affinity to the ionophore but does not support activation, did not suppress the response to the ionophore. Similarly, Mn++ which binds to the ionophore with a 100-fold higher affinity than Ca++ was not able to interfere with the activation of lymphocytes. Both ions, however, inhibited the ionophore-dependent Ca++ influx at concentrations not affecting activation. Within 1 hr of contact the ionophore increased the turnover of the phospholipid fatty acids. The dose response curve for the activation of the membrane phospholipid metabolism and that for the stimulation of RNA or DNA synthesis were identical. These data suggest that the ionophore A 23 187 interacts directly with rabbit (B) lymphocytes to induce cell activation. The activating capacity does not depend on the translocation of Ca++. We propose that the ionophore intercalates into the lipid phase of the plasma membrane, initiating the same changes in the membrane itself that are induced by mitogens that bind to surface receptors, i.e., increasing the turnover of membrane phospholipids.