Cell Regulatory Molecules Research Articles

Cross-Regulation of iNOS and COX-2 by its Products in Murine Macrophages Under Stress Conditions

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43°C, 30min) and stimulated with LPS (1 µg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE₂ by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE₂ and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37°C. These treatments significantly reduced MTT levels in cells at 37°C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.

Future molecular aspects of cervical cytology

Summary Cervical cancer, a potentially preventable disease, remains the second most common malignancy in women worldwide. Current screening protocols rely on the Pap smear test, which has a reported false-negative rate of 15–50%. Advances in automation of cervical cytology have resulted in improved cell preservation techniques and overall high-quality cellular material that is suitable for molecular analysis. The current focus is primarily on the use of molecular biomarkers as adjuncts to existing screening procedures. These biomarkers include human papillomavirus (HPV) and the host cell regulatory molecules, minichromosome maintenance proteins, Cdc6 (a cell division cycle protein) and p16(INK4A) tumour suppressor protein. Developments in micro-array technology and its application to the study of cervical cancer have greatly expanded the list of differentially regulated genes known to be involved in cervical cancer. This will help to unravel the pathogenesis of HPV infection and dysplastic progression, and ultimately improve treatment of cervical intra-epithelial neoplasia and cervical cancer.

Ras‐Associated Small GTPase 33A, a Novel T Cell Factor, Is Down‐Regulated in Patients with Tuberculosis

Ras-associated small GTPases (Rabs) are specific regulators of intracellular vesicle trafficking. Interference with host cell vesicular transport is a hallmark of many intracellular pathogens, including the notable example Mycobacterium tuberculosis. We performed, by quantitative polymerase chain reaction, gene-expression analyses for selected Rab molecules in peripheral-blood mononuclear cells from patients with tuberculosis (TB) and healthy control subjects, to identify candidate genes that are critically involved in the host immune response. Comparison revealed significant differences in the expression of genes for Rab13, Rab24, and Rab33A. Rab33A gene expression was down-regulated in patients with TB and was predominantly expressed in CD8+ T cells. We excluded possible influences of differences in T cell percentages between the 2 study groups, demonstrating that Rab33A gene expression changes on the single-cell level. In vitro, Rab33A RNA expression was induced in T cells on activation and by dendritic cells infected with M. tuberculosis. Our findings identify Rab33A as a T cell regulatory molecule in TB and suggest its involvement in disease processes.

CTLA-4 regulates the murine immune response to Trypanosoma cruzi infection.

Infection with Trypanosoma cruzi causes a profound suppression of T cell responsiveness to polyclonal or antigenic stimuli. In this study, we quantified expression of the negative T cell regulatory molecule CTLA-4 in T. cruzi infected mice and analysed its influence on the immune suppression. Levels of splenic CTLA-4 expression were highest around day 10 after infection, reaching 5% in resistant B6D2F1 mice, but exceeding 10% of CD4(+) T cells in C57BL/6 mice that were susceptible to mortal disease. The proliferative response of explanted splenocytes to CD3-mediated stimulation was strongly suppressed in both the susceptible and the resistant strains. Blockade of CTLA-4 in vitro with a monoclonal antibody affected neither proliferative response nor cytokine production (IFN-gamma, IL-4 and IL-2) by splenic T cells from infected C57BL/6 mice. Treatment of mice with anti-CTLA-4 antibody on the day of infection decreased IFN-gamma production and reduced mortality by about 50%. We conclude that high CTLA-4 expression is a hallmark of severe disease in murine T. cruzi infection, and that CTLA-4 has a regulative influence at the early stages during priming of the immune reaction to the parasite, augmenting a strong Th1-biased response.

Assaying protein kinase activity.

Protein kinases, encoded by approx 2% of eukaryotic genes, represent one of the major classes of cell-regulatory molecules. Assessment of the catalytic activity of a specific protein kinase can be an important step in elucidating signal-transduction pathways that affect cell behavior. As an example of approaches taken to measure protein kinase activity, this chapter presents methods useful for determination of the activity of the oncogenic protein-tyrosine kinase v-Src. Included are protocols for heterologous expression of the kinase in yeast Saccharomyces cerevisiae, immunoaffinity purification from yeast cell lysates, kinase reactions using incorporation of 32P into peptide substrates, and quantifying protein kinase activity. The Notes section discusses alternative approaches for assaying the activity of Src recovered from vertebrate cells and it gives recommendations for assaying the activity of the other protein kinases with respect to the substrate specifity and the composition of kinase reaction buffer.

Cutting edge: molecular analysis of the negative regulatory function of lymphocyte activation gene-3.

Lymphocyte activation gene (LAG)-3 (CD223) is a CD4-related activation-induced cell surface molecule that binds to MHC class II molecules with high affinity and negatively regulates T cell expansion and homeostasis. In this study, we show that LAG-3 inhibits CD4-dependent, but not CD4-independent, T cell function via its cytoplasmic domain. Although high affinity interaction with MHC class II molecules is essential for LAG-3 function, tailless LAG-3 does not compete with CD4 for ligand binding. A single lysine residue (K468) within a conserved "KIEELE" motif is essential for interaction with downstream signaling molecules. These data provide insight into the mechanism of action of this important T cell regulatory molecule.

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I

Articular cartilage is routinely subjected to mechanical forces and to cell-regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin-like growth factor-I (IGF-I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF-I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF-I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF-I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF-I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF-I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF-I and increased transport of IGF-I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.

Searching for stem cell regulatory molecules. Some general thoughts and possible approaches.

Hematopoietic development in the mammal can be represented as a numerically expanding hierarchy of cell populations that are progressively restricted in their self-renewal and differentiation abilities. Classical functional studies have now been extended to provide exact physical descriptions of various stages in the hematopoietic hierarchy. In particular, much information is available that defines the properties of the most primitive stem cell compartment. In addition, a number of in vitro culture systems suggest the possibility of maintaining and expanding these cells in a defined context. In all developmental systems, unique profiles of expressed genes define distinct differentiation stages. Within these profiles are gene products that play crucial roles in the regulation of cell-fate decisions. Recent progress in hematopoietic biology provides the framework within which to define molecular phenotypes for hematopoietic stem cells and their immediate clonal progeny. Identifying novel gene products expressed predominantly in uncommitted stem cells together with functional loss and gain-of-function approaches should begin to unravel the molecular mechanisms that govern biological phenomena such as self-renewal, commitment, and proliferation in the hematopoietic system.

CTLA-4 Regulates Tolerance Induction and T Cell Differentiation In Vivo

Cytotoxic T lymphocyte Ag-4 (CTLA-4; CD152) is an important T cell regulatory molecule. In vitro experiments have shown that the blockade of signals through CTLA-4 augments T cell expansion, while CTLA-4 cross-linking results in decreased T cell proliferation due to decreased IL-2 production. However, less is known about the role of CTLA-4 in regulating an ongoing immune response. In this study, we examined the role of CTLA-4 in the expansion, decline, tolerization, and differentiation of T cells following treatment with staphylococcal enterotoxin B (SEB). Anti-CTLA-4 treatment resulted in increased numbers of SEB-reactive T cells and blockade of subsequent tolerance induction. Further examination of the SEB-reactive cells from anti-CTLA-4-treated mice demonstrated that both the CD4+ and CD8+ Vbeta8+ T cells produced IL-4, providing evidence that not only do signals through CTLA-4 regulate T cell-tolerizing events, but they also play an important role in the differentiation of T cells in vivo.

A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium.

A number of bacterial pathogens have evolved sophisticated strategies to subvert host-cell signal-transduction pathways for their own benefit. These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells. In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence. Sequence analysis revealed that a carboxy-terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases. Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine. An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases. In addition, an amino-terminal region of SptP exhibited sequence similarity to the ribosyltransferase exoenzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp. The modular nature of this effector protein may allow multiple interactions with host-cell signalling functions.

Cell adhesion and regulatory molecules involved in tumor formation, hemostasis, and wound healing.

Dynamic changes in cell adhesion and migration are fundamental properties of the cells that are important in hemostasis, vascularization, and wound repair. Changes in cell adhesion and migration are very important in the formation of tumors, and invasion and metastasis by neoplasms. Using immunolocalization and immunopurification techniques, expression of integrin molecules involved in cell adhesion has been characterized during tumor development and wound healing. The ability of cytokines to regulate expression and antibody and peptide inhibitors to inhibit function of integrins has been explored to better understand the role of integrins in pathogenesis. Increased suprabasilar expression of the integrins alpha 6 beta 4, alpha 2 beta 1, and alpha 3 beta 1 accompanies development of squamous cell neoplasms of the head and neck. Integrin alpha II beta 3 is an integrin receptor which is important in platelet aggregation in hemostasis and formation of tumor metastases. Increased expression of integrin alpha v beta 3 has been identified in angiogenesis in response to growth factors and tumor angiogenic factors. Cytokine TGF-beta and arachadonic acid metabolite 12(S)-HETE are important signals in regulation of integrins and affect cell aggregation and migration in these processes. Ligands and inhibitors for some of these receptors have been identified which will be useful in determining their role during tumor development and progression. Recent advances in understanding the functions of the integrin cell adhesion molecules may soon add new diagnostic methods and therapies to the armamentarium of the head and neck oncologic, microvascular, plastic, and reconstructive surgeon. Therapies directed at controlling integrin cell-adhesion molecule expression and function are being explored to improve scar formation, bone synthesis, inhibition of vascular occlusion, and inhibition of tumor invasion and metastasis.

Leukocyte interaction with protein cascades in blood coagulation.

Coagulation preserves the homeostasis of internal body fluids against life-threatening blood losses. Although generally viewed as a regulated enzymatic cascade, this mechanism depends on the participation of vascular cells. Leukocytes, in particular, have evolved a formidable machinery to initiate and amplify blood clotting reactions through the recognition of cell surface receptors, proteolytic enzymes, and cofactor and regulatory molecules. The consequences of thrombin generation on the leukocyte surface are not exclusively restricted to the hemostatic balance. Rather, thrombin and other coagulation proteases influence a panoply of cellular functions, ranging from activation pathways and adherence phenomena, to DNA synthesis and cell proliferation of normal and malignant phenotypes. For this multivalency of cellular responses, the process of blood coagulation is now perceived as a broad cell signaling mechanism that participates in all pathophysiological aspects of host inflammatory responses.

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor.

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)

Interferons as cell-regulatory molecules

This review presents the current evidence for interferons as cell-regulatory molecules. Apart from inducing an antiviral state, interferon preparations are powerful inhibitors of cell growth and have selective effects on cellular protein synthesis. In addition, interferons are produced during most immune reactions and can exert positive and negative influences on these reactions. Thus interferon molecules are of interest to cell biologists, immunologists, and oncologists. Interferon as a cell regulator offers a unique approach to cancer therapy, but for its judicious use, more understanding of basic mechanisms of action is required.