Suppression Of Cellular Responses Research Articles

Activation of Transient Receptor Potential Ankyrin-1 (TRPA1) in Lung Cells by Wood Smoke Particulate Material

Cigarette smoke, diesel exhaust, and other combustion-derived particles activate the calcium channel transient receptor potential ankyrin-1 (TRPA1), causing irritation and inflammation in the respiratory tract. It was hypothesized that wood smoke particulate and select chemical constituents thereof would also activate TRPA1 in lung cells, potentially explaining the adverse effects of wood and other forms of biomass smoke on the respiratory system. TRPA1 activation was assessed using calcium imaging assays in TRPA1-overexpressing HEK-293 cells, mouse primary trigeminal neurons, and human adenocarcinoma (A549) lung cells. Particles from pine and mesquite smoke were less potent agonists of TRPA1 than an equivalent mass concentration of an ethanol extract of diesel exhaust particles; pine particles were comparable in potency to cigarette smoke condensate, and mesquite particles were the least potent. The fine particulate (PM < 2.5 μm) of wood smoke were the most potent TRPA1 agonists and several chemical constituents of wood smoke particulate, 3,5-ditert-butylphenol, coniferaldehyde, formaldehyde, perinaphthenone, agathic acid, and isocupressic acid, were TRPA1 agonists. Pine particulate activated TRPA1 in mouse trigeminal neurons and A549 cells in a concentration-dependent manner, which was inhibited by the TRPA1 antagonist HC-030031. TRPA1 activation by wood smoke particles occurred through the electrophile/oxidant-sensing domain (i.e., C621/C641/C665/K710), based on the inhibition of cellular responses when the particles were pretreated with glutathione; a role for the menthol-binding site of TRPA1 (S873/T874) was demonstrated for 3,5-ditert-butylphenol. This study demonstrated that TRPA1 is a molecular sensor for wood smoke particulate and several chemical constituents thereof, in sensory neurons and A549 cells, suggesting that TRPA1 may mediate some of the adverse effects of wood smoke in humans.

Cancer cells utilize a novel regulatory subset of B cells to promote escape and metastasis (100.26)

Abstract B cells play an important role in antigen presentation and antibody production. However, they appear to exhibit suppressive function inhibiting autoimmune responses in mice. Yet, it remains unknown whether regulatory B cells exist or participate in cancer progression. Here, we demonstrate that a unique B cel subset is induced from resting B cells by cancer cells. These B cells, designated as tumor Bregs (tBregs), are poorly proliferative and express high level of IL-2Rα. The major function of tBregs is to suppress T cell responses, as they efficiently and in dose dependent manner inhibits a proliferation of T cells stimulated with anti-CD3/28 beads. The suppressive activity of tBregs is a cell contact-dependent, but Fas/FasL- and PD1/B7H1-independent, and unlike any other reported regulatory cells, does not require soluble factors such as IL-2, IL-10, TGFβ, IL-27 and IL-35. Importantly, we show for the first time, tBregs induce a conversion of Tregs from non-regulatory CD4+ T cells. Taken together, tBregs are an important and distinct subset of regulatory cells which promote cancer escape and metastasis through direct and indirect (via Tregs) suppression of cellular responses. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.

Evasion of the Interferon-Mediated Antiviral Response by Filoviruses

The members of the filoviruses are recognized as some of the most lethal viruses affecting human and non-human primates. The only two genera of the Filoviridae family, Marburg virus (MARV) and Ebola virus (EBOV), comprise the main etiologic agents of severe hemorrhagic fever outbreaks in central Africa, with case fatality rates ranging from 25 to 90%. Fatal outcomes have been associated with a late and dysregulated immune response to infection, very likely due to the virus targeting key host immune cells, such as macrophages and dendritic cells (DCs) that are necessary to mediate effective innate and adaptive immune responses. Despite major progress in the development of vaccine candidates for filovirus infections, a licensed vaccine or therapy for human use is still not available. During the last ten years, important progress has been made in understanding the molecular mechanisms of filovirus pathogenesis. Several lines of evidence implicate the impairment of the host interferon (IFN) antiviral innate immune response by MARV or EBOV as an important determinant of virulence. In vitro and in vivo experimental infections with recombinant Zaire Ebola virus (ZEBOV), the best characterized filovirus, demonstrated that the viral protein VP35 plays a key role in inhibiting the production of IFN-α/β. Further, the action of VP35 is synergized by the inhibition of cellular responses to IFN-α/β by the minor matrix viral protein VP24. The dual action of these viral proteins may contribute to an efficient initial virus replication and dissemination in the host. Noticeably, the analogous function of these viral proteins in MARV has not been reported. Because the IFN response is a major component of the innate immune response to virus infection, this chapter reviews recent findings on the molecular mechanisms of IFN-mediated antiviral evasion by filovirus infection.

An experimental model for the study of drug effects on cutaneous healing

Immunosuppressive agents are known to interfere with the healing of surgical wounds. The increasing use of these drugs warrants a better understanding of their effects on wound healing. The aim of this study was to develop an experimental model that would allow for a reliable and rapid assessment of drug effects on cutaneous wound healing. Thirty syngeneic Lewis rats underwent surgical incision on their dorsal region, in the presence or absence of a three-week regimen of immunosuppressant drug therapy (i.e., cyclosporin, azathioprine, and prednisone). Surgical site tissue was collected at intervals over 21 days after surgery and analyzed for cell number and collagen fiber content. Both of these quantitative assessments were performed using digital image capture with the Image Pro Plus 4.5 software. Computerized histomorphometric analyses revealed an apparent inhibition of cellular responses and collagen fiber production in drug-treated animals compared to control animals. The experimental model was reproducible, easy to perform, and allowed for quantitative histological evaluations. It may be useful for the study of surgical healing in the presence of other drug classes.

Reactive oxygen species mediates disialoganglioside GD3‐induced inhibition of ERK1/2 and matrix metalloproteinase‐9 expression in vascular smooth muscle cells

Sialic acid containing glycosphingolipids (gangliosides) are thought to play important roles in the function of various biological phenomena such as atherosclerosis. We have previously shown that the overexpression of the disialoganglioside (GD3) synthase gene effectively suppresses cell proliferation, cell cycle progression, and MMP-9 expression in vascular smooth muscle cells (VSMC). However, the issue of how the overexpression of GD3 synthase gene results in the inhibition of cellular responses in VSMC remains unclear. The findings herein demonstrate that overexpression of the GD3 synthase gene suppresses VSMC responses through the generation of reactive oxygen species (ROS). Superoxide and hydrogen peroxide were generated at increased levels in GD3 synthase gene transfectants in comparison with empty vector (EV) -transfected VSMC. This phenomenon was blocked by antioxidants such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Increased ROS generation was associated with a decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Further studies revealed that the blockade of ROS function with antioxidants reversed the effect of GD3 synthase gene overexpression on VSMC proliferation and cell cycle regulation in response to platelet-derived growth factor (PDGF). In addition, we found that treatment with antioxidants reversed the decreased matrix metalloproteinase-9 (MMP-9) expression in response to TNF-alpha as determined by zymography and immunoblot in GD3 synthase gene transfectants. This recovery effect was characterized by the up-regulation of MMP-9 promoter activity, which was transcriptionally regulated at NF-kappaB and activation protein-1 (activating protein (AP) -1) sites in the MMP-9 promoter. These findings suggest that ROS may play a role in GD3 synthase gene-mediated VSMC phenotypic changes that may contribute to plaque instability in atherosclerosis.

Cross-Antagonism of a T Cell Clone Expressing Two Distinct T Cell Receptors

Inhibition of T cell activation can be mediated by analogs of the original antigenic peptide (TCR antagonists). Here, a T cell clone expressing two distinct TCR was used to investigate whether such inhibition involves an active mechanism by examining whether an antagonist for one TCR could influence responses stimulated by the other TCR engaging its agonist. Our results demonstrate functional cross-inhibition under these conditions involving the ability of antagonist:TCR interactions to diminish Lck enzymatic activity associated with the agonist-recognizing second TCR, apparently through enhancement of SHP-1 association with these receptors. Our findings reveal that inhibition of cellular responses by antagonists arises at least in part from active negative regulation of proximal TCR signaling and identify elements of the biochemical process.

Defect in potentiation of adenylyl cyclase correlates with bronchial hyperreactivity

Background: Adenylyl cyclase is a transmembrane signaling system involved in the inhibition of cellular responses. Recently, we showed that the activity of adenylyl cyclase may be potentiated by stimuli that induce an increase of cellular responses but that do not activate adenylyl cyclase. This is probably an important physiologic feedback mechanism that prevents cells from becoming “overstimulated.” Objective: Because increased cellular activities are frequently observed in persons with asthma, we hypothesized that a defect in potentiation of adenylyl cyclase might be involved. Methods: Potentiation of isoprenaline-induced adenosine cyclic monophosphate (cAMP) production with the mitogen phytohemagglutinin (PHA; 45 μg/ml) or the calcium ionophore A23187 (1 μmol/L) was studied in peripheral blood mononuclear cells taken from patients with asthma ( n = 8) and healthy control subjects ( n = 11). Results: Isoprenaline-induced cAMP production was potentiated significantly in the healthy control subjects (PHA, 110% ± 15%; A23187, 92% ± 25%). In contrast, potentiation was not seen with PHA or A23187 in the total group of patients with asthma. However, some patients showed weak potentiation, whereas in others PHA decreased isoprenaline-induced cAMP production. Moreover, the effect of PHA on isoprenaline-induced cAMP production correlated significantly with the degree of bronchial hyperreactivity in patients with asthma ( r = 0.96; p = 0.0001). Conclusions: The observed defect in signal transduction could play an important part in bronchial hyperresponsiveness. (J A LLERGY C LIN I MMUNOL 1995;96:628-34.)

Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response.

Staphylococcus epidermidis infection of plastic catheters is often associated with heavy deposits of slime. To test whether this slime affects the human cellular immune response, its effect on the lympho-proliferative response of mononuclear cells to polyclonal stimulators was measured. Slime drastically reduces this response. Its inhibitory action was not immediate but took place over a few days and resulted in destruction of affected cells. The effect is dose related. This inhibition of cellular response may contribute to S epidermidis infection of implanted prostheses.

Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid.

Mepacrine has been used as an inhibitor of the activation of endogenous phospholipases in many systems. These endogenous phospholipases are important in the modification of the lipid environment of membrane proteins and in the release of locally active oxygenated arachidonic acid metabolites. In both human platelets and erythrocytes, mepacrine blocks the release of fatty acid from phospholipid by endogenous phospholipases. However, mepacrine also interacts directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives. This interaction occurs rapidly and is maximal at concentrations of mepacrine greater than 0.2 mM. Such drug-phospholipid interaction may perturb membrane architecture and function and be responsible for the inhibitory effects of mepacrine on cellular responses observed in many systems. Since the alteration in membrane phospholipid composition occurs under the same conditions as phospholipase inhibition, it is not possible to be certain that the inhibition of cellular responses by mepacrine is due to inhibition of phospholipases rather than to direct perturbation of the membrane. It is also possible that inhibition of phospholipase action by mepacrine is in part a consequence of the change in phospholipid composition. These results indicate that caution should be exercised in the interpretation of results obtained using mepacrine and that the usefulness of this compound for the investigation of the biological importance of phospholipase activation is limited.

Suppression of cellular responses in mice during Trypanosoma cruzi infections.

Immunosuppression has been reported to occur in several protozoan parasitic infections. The significance of this suppression on host resistance or on parasite avoidance of immune destruction has not, however, been determined. In the present study two strains of mice that differ with respect to resistance to Trypanosoma cruzi were examined during the course of infection for differences in expression of suppression of blastogenic responses to phytohemagglutinin and an antigen preparation of these parasites. It was found that in vitro blastogenic responses were suppressed in both strains of mice: on day 12 for C57BL/6 mice (resistant strain) and on day 17 for C3H(He) mice (susceptible strain). Neither C3H(He) nor C57BL/6 lymph node cells (LNC) responded to a crude sonically treated antigen of these parasites, although C57BL/6 LNC were inhibited by this antigen later in infection. There was no abrogation of suppression of LNC responses late in infection, when decreases in spleen weight or total spleen cell numbers occurred, in the resistant C57BL/6 mice. LNC from normal uninfected mice were found to be completely suppressed in responsiveness to phytohemagglutinin when cultured at a 2:1 ratio with LNC from C57BL/6 mice with 18-day infections. Attempts to characterize the cell type responsible for this suppression showed it to be a non-thy 1.2-bearing, nylon wool-adherent cell.