IFN-gamma reduces specific binding of tumor necrosis factor on murine macrophages.

Because IFN-gamma is the main cytokine activating macrophages and TNF cooperates in this activation, we assessed TNF binding capacity during the course of murine macrophage activation by IFN-gamma. TNF binding to elicited macrophages increased with time, was maximal by 8 h of culture, and required de novo protein synthesis. 125I-TNF bound to about 40,000 sites/cell with a Kd of 1 x 10(-9) M. Cross-linking experiments performed with a bifunctional cross-linking agent revealed a specific band with a m.w. of 94,000. Preincubation of macrophages with IFN-gamma prevented the binding of TNF to receptors. This effect was dose-dependent and maximal at 100 U/ml. IFN-gamma also reduced specific TNF binding to preexisting receptors (50% inhibition in 3 h), but IFN-gamma did not change the internalization rate of TNF. These studies showed that the number of TNF receptors increased on macrophages vs maturation in culture and was negatively controlled by IFN-gamma.     

Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.

Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.     

Effects of recombinant murine tumor necrosis factor-alpha on immune function

TNF-alpha is a macrophage-derived cytokine with diverse biologic activities, including potent immunomodulatory effects. In vitro studies have implied that TNF-alpha has predominantly proinflammatory and immunostimulatory effects, but paradoxically in vivo studies have demonstrated that administration of TNF-alpha suppresses murine lupus. To assess the effects of TNF-alpha on immune function in normal mice, we treated C57BL/6 mice with recombinant murine TNF-alpha (10 micrograms i.p.) or PBS on alternate days for up to 8 wk. Administration of TNF-alpha decreased the percentage of splenic T and B cells and increased the percentage of splenic macrophages without significantly altering the total number of mononuclear cells. Administration of TNF-alpha also caused progressive inhibition of splenic lymphocyte function, out of proportion to the quantitative reduction in B and T cells. After 8 wk of therapy, the proliferative responses of splenic lymphocytes to Con A, PHA, and LPS were reduced by 100, 90, and 60%, respectively, in treated mice compared with control mice. The reduction in T cell proliferation was due primarily to alteration of accessory cell function rather than direct inhibition of T cell function. Treatment with TNF-alpha markedly inhibited T cell cytotoxicity induced by immunization with allogenic target cells, and it virtually ablated NK cell activity. Inhibition of these in vitro tests of lymphocyte function correlated with inhibition of delayed type hypersensitivity in vivo. In contrast, treatment with TNF-alpha did not impair humoral immunity. These findings imply that TNF-alpha may affect cell-mediated immunity more profoundly than humoral immunity. This observation may be relevant to the mechanism whereby TNF-alpha suppresses murine lupus.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.     

Overexpression of tumor necrosis factor-alpha increases production of hydroxyl radical in murine myocardium

Transgenic (TG) mice with cardiac-specific overexpression of tumor necrosis factor-alpha develop congestive heart failure with myocardial inflammation. The purpose of this study was to investigate the effects of tumor necrosis factor-alpha on reactive oxygen species (ROS) in this mouse model of cardiomyopathy. Myocardial production of hydroxyl radical detected by electron spin resonance spectroscopy was significantly increased in TG. Myocardial expression of Mn-SOD was significantly decreased in TG, whereas that of Cu,Zn-SOD was unaltered. Myocardial expression of catalase was unchanged, whereas that of glutathione peroxidase was significantly increased, in TG. Histological analysis revealed that macrophages and CD4-positive lymphocytes were increased in TG myocardium. To investigate whether these infiltrating inflammatory cells were the source of ROS, we treated TG mice with cyclophosphamide for 7 days. Although cyclophosphamide significantly suppressed the infiltration of inflammatory cells, it did not diminish the production of hydroxyl radical in TG myocardium. Damaged myocytes, but not infiltrating inflammatory cells, may be the source of ROS in TG.     

Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.     

IFN-gamma, tumor necrosis factor-alpha, and urokinase regulate the expression of urokinase receptors on human monocytes

The ability of macrophages to reach inflammatory loci is crucial in the function of cellular immunity. Invasive properties of macrophages may be due to the proteinase urokinase which binds to cell surface receptors, and thereby confers on macrophages the capacity for localized proteolysis of the interstitium. Here, we investigated the role of the macrophage-activating factors IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF and of urokinase on the expression of urokinase receptors by human cultured monocytes. IFN-gamma and TNF-alpha induced increased urokinase binding to human cultured monocytes in a time- and dose-dependent fashion. At optimal concentrations, IFN-gamma (200 U/ml) increased the number of receptors/cell from 14,000 to 64,000, TNF-alpha (50 U/ml) to 30,000, and combinations of IFN-gamma and TNF-alpha to 90,000. Granulocyte-macrophage-CSF had no effect. The enhanced urokinase binding is due to increased numbers of urokinase receptors and not an increased affinity of the receptor for urokinase. In the presence of urokinase during monocyte activation, IFN-gamma induced only 25,000 receptors/cell. However, urokinase does not inhibit increased receptor expression when the cells are activated with TNF-alpha. The effect of urokinase on induction of urokinase receptors by combinations of IFN-gamma and TNF-alpha varied with the dosage of TNF-alpha: A combination of IFN-gamma (200 U/ml) and TNF-alpha (15 U/ml) induced 38,000 receptors/cell in the presence and 90,000 receptors/cells in the absence of urokinase, whereas IFN-gamma (200 U/ml) and TNF-alpha (20 U/ml) induced 90,000 receptors/cell in the absence and presence of urokinase. These studies demonstrate that IFN-gamma, TNF-alpha, and urokinase collectively regulate the number of urokinase receptors on human monocytes. The induction of urokinase receptors may be responsible for increased invasiveness of the activated macrophage.     

The effect of fluconazole treatment on tumor necrosis factor-alpha production in murine candidiasis

That systemic candidiasis increases TNF-alpha has been proved in various studies. In our study, we investigated if fluconazole too, which is used in treatment, has an additional effect on the increase of TNF-alpha. On the sixth day of the experimental infection, it was measured that TNF-alpha levels of candidiasis was 699 +/- 60 pg/ml whereas it was 683 +/- 35 pg/ml in the mice that were treated with fluconazole. TNF-alpha levels were measured by enzyme-linked immunosorbant assay. Due to the fact that no statistically significant difference was found between them (p > .05) it was thought that fluconazole did not have an additional effect on the increase of TNF-alpha in candidiasis.     

Modulation of granulocyte colony-stimulating factor receptors on murine peritoneal exudate macrophages by tumor necrosis factor-alpha.

Modulation of granulocyte CSF (G-CSF) receptors on murine peritoneal exudate macrophages (PEM) by various cytokines was investigated. At 4 degrees C, 125I-G-CSF receptor binding on PEM reached a plateau after 6 h and was specifically competed by unlabeled human rG-CSF but not by other cytokines, including human rG-CSF-1, murine recombinant granulocyte-macrophage CSF, murine rIFN-gamma, human rIL-1 beta, and murine rTNF-alpha. 125I-G-CSF bound to PEM has a half-life of 30 min at 37 degrees C. Preincubation of PEM with murine rTNF, murine recombinant granulocyte-macrophage CSF, CSF-1, or G-CSF for 30 min at 37 degrees C resulted in partial reduction of 125I-G-CSF binding capacity, whereas IL-1 or IFN-gamma did not inhibit G-CSF binding. Further studies indicated that reduction of G-CSF binding caused by TNF was a dose- and time-dependent process and did not require FCS. The reduction was transient, and receptor binding was recovered by incubation at 37 degrees C for 8 h. The recovery of G-CSF binding was inhibited in the presence of cycloheximide. In addition, G-CSF binding studies suggested that the TNF-induced decrease in G-CSF binding to PEM was probably due to a reduction in receptor number rather than receptor affinity. Modulation of G-CSFR by TNF was also observed on nonelicited macrophages from various strains of mice. Our results demonstrate a physiologic response of G-CSFR on macrophages that is modulated by TNF. This phenomenon may play an important, as yet unknown, role in the macrophage inflammatory response.     

Cytotoxic mechanism of tumor necrosis factor-alpha

Many intracellular pathways are set in motion by the binding of tumor necrosis factor (TNF) to its cell surface receptor. Major steps in the TNF-mediated cytotoxicity cascade include G protein-coupled activation of phospholipases, generation of free radicals, and damage to nuclear DNA by endonucleases. Ultimately the cells undergo apoptosis and die. Understanding how TNF initiates these pathways will facilitate the rational design of pharmaceuticals that can attenuate or potentiate the action of this important cytokine.     

IFN-gamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha.

Activated murine peritoneal macrophages inhibit the intracellular proliferation of Toxoplasma gondii and produce a number of cytokines, such as TNF-alpha and IL-1. Both TNF-alpha and IL-1 have been reported to be involved in the immune response against various microorganisms, but the mechanisms responsible for these effects are not known. In the present study it was investigated whether endogenously produced TNF-alpha and IL-1 are involved in the activation of peritoneal macrophages by rIFN-gamma leading to toxoplasmastatic activity and the production of reactive nitrogen intermediates. The rIFN-gamma-induced toxoplasmastatic activity was inhibited by neutralizing antibodies against mouse TNF-alpha in a dose-dependent and time-dependent way, but neutralizing antibodies against mouse IL-1 alpha and IL-1 beta did not affect this activity. Involvement of TNF-alpha in the induction of toxoplasmastatic activity was confirmed by our finding that rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma inhibited the intracellular proliferation of T. gondii. No synergistic activity of rIL-1 and rIFN-gamma on the inhibition of T. gondii proliferation was found. Both rTNF-alpha and rIL-1 alpha alone inhibited the intracellular proliferation of T. gondii only slightly. Because it has been reported recently that activated macrophages produce reactive nitrogen intermediates that are essential in the induction of toxoplasmastatic activity, we investigated whether these intermediates are involved in the TNF-dependent induction of toxoplasmastatic activity. Neutralizing antibodies against mouse TNF-alpha inhibited also the release of NO2- by rIFN-gamma-activated macrophages almost completely. Macrophages incubated with rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma released substantial amounts of NO2-, but rTNF-alpha and rIL-1 alpha alone, and the combination of rIL-1 alpha and a nonactivating concentration of rIFN-gamma induced only little NO2(-)-release by macrophages. To assess whether reactive nitrogen intermediates act directly or indirectly on the intracellular proliferation of T. gondii, macrophages were incubated with the L-arginine analog NG-monomethyl-L-arginine or the NADPH-inhibitor diphenylene iodonium, both inhibitors of the generation of reactive nitrogen intermediates. Good correlation was found between toxoplasmastatic activity and the release of NO2- during the 24-h activation period before infection of the macrophages with T. gondii, but no correlation was found between toxoplasmastatic activity and the release of NO2- during infection of the macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

Molecular design of conjugated tumor necrosis factor-alpha: synthesis and characteristics of polyvinyl pyrrolidone modified tumor necrosis factor-alpha

We conjugated tumor necrosis factor-alpha (TNF-alpha) with the synthetic polymeric modifier polyvinyl pyrrolidone (PVP) to facilitate its clinical use for anti-tumor therapy. TNF-alpha was chemically conjugated with the terminal carboxyl-bearing PVP at one end of its main chain, which was radically polymerized via the formation of an amide bond between the lysine amino groups of TNF-alpha and carboxyl group of PVP. In vitro specific bioactivity of PVP-conjugated TNF-alpha (PVP-TNF-alpha) relative to that of native TNF-alpha gradually decreased with increases in the degree of PVP attachment. In contrast, PVP-TNF-alpha in which 40% of TNF-alpha lysine residues were coupled with PVP (MPVP-TNF-alpha) exhibited the highest anti-tumor activity among the conjugated derivatives examined. MPVP-TNF-alpha had more than 200-fold higher anti-tumor efficacy than native TNF-alpha, and the anti-tumor activity of MPVP- TNF-alpha was more than 5-fold stronger than that MPEG- TNF-alpha which had the highest anti-tumor activity among PEG-conjugated TNF-alphas examined. Additionally, a high dose of native TNF-alpha induced toxic side-effects such as body weight reduction, piloerection and tissue inflammation, while no side effects were observed following i.v. administration of MPVP-TNF-alpha. The plasma half-life of MPVP-TNF-alpha (360 min) was about 80 and 3-fold longer than those of native TNF-alpha (4.6 min) and MPEG-TNF-alpha (122 min), respectively. These results suggested that PVP is a useful polymeric modifier for increasing the anti-tumor activity of PVP.     

Specific release of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IFN-gamma by human tumor-infiltrating lymphocytes after autologous tumor stimulation

Tumor-infiltrating lymphocytes (TIL) have been cultured from a variety of human tumors, and some melanoma TIL have demonstrated specific, MHC-restricted recognition of autologous tumor in short term lysis assays. The current study investigates cytokine release by TIL as an indicator of specific tumor recognition. We have identified two of four melanoma and one of seven breast carcinoma TIL cultures that specifically release granulocyte-macrophage-CSF, TNF-alpha, and IFN-gamma after autologous tumor stimulation. The other cultures either do not secrete cytokine or secrete cytokine in a nonspecific fashion. The amount of specific cytokine released is directly related to the number of TIL and stimulating tumor cells. Studies of TIL, from two melanoma patients, separated into CD4+ and CD8+ populations revealed that CD8+ cells were responsible for virtually all of the specific cytokine secretion, although both populations released cytokines when activated by immobilized anti-CD3 antibody. Specific cytokine release by CD8+ TIL was inhibited by anti-MHC class I mAb. Specific cytokine release was also detected from a CD4+ breast cancer TIL culture, and this was inhibited by anti-MHC class II mAb. The clinical significance of this specific mode of immune antitumor reactivity is currently under investigation.     

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.     

Shedding of ICAM-1 from human melanoma cell lines induced by IFN-gamma and tumor necrosis factor-alpha. Functional consequences on cell-mediated cytotoxicity.

ICAM-1-mediated cell-cell adhesion is essential for various immunologic functions, including non-MHC-restricted cytotoxicity. The present study was designed to establish whether shedding of ICAM-1 from melanoma cells occurred and to characterize the effects of soluble ICAM-1 on some cell adhesion-dependent functions. The shed soluble ICAM-1 molecule was detected and quantified by a specific ELISA. Shedding of ICAM-1 could be induced by IFN-gamma and TNF-alpha alone, or more effectively, by a combination of the two cytokines together. The use of purified soluble ICAM-1 enabled us to test for the functional significance of the ICAM-1 shedding from tumor cells. Conjugate formation between the cloned NK cell line CNK6 and the erythromyeloid cell line K562, as well as between lymphokine-activated killer cells and the melanoma cell line M26, could be inhibited by purified soluble ICAM-1 and cell-free supernatants from melanoma cell cultures containing shed ICAM-1. Furthermore, the non-MHC-restricted cytotoxicity mediated by NK and lymphokine-activated killer cells could be abrogated either by purified soluble ICAM-1 or by melanoma cell culture supernatants containing shed ICAM-1. Thus, shedding of ICAM-1 may be one of the mechanisms by which neoplastic cells escape immunosurveillance.     

Eicosanoids and tumor necrosis factor-alpha in the kidney

The thick ascending limb of Henle's loop (TAL) is capable of metabolizing arachidonic acid (AA) by cytochrome P450 (CYP450) and cyclooxygenase (COX) pathways and has been identified as a nephron segment that contributes to salt-sensitive hypertension. Previous studies demonstrated a prominent role for CYP450-dependent metabolism of AA to products that inhibited ion transport pathways in the TAL. However, COX-2 is constitutively expressed along all segments of the TAL and is increased in response to diverse stimuli. The ability of Tamm-Horsfall glycoprotein, a selective marker of cortical TAL (cTAL) and medullary (mTAL), to bind TNF and localize it to this nephron segment prompted studies to determine the capacity of mTAL cells to produce TNF and determine its effects on mTAL function. The colocalization of calcium-sensing receptor (CaR) and COX-2 in the TAL supports the notion that activation of CaR induces TNF-dependent COX-2 expression and PGE? synthesis in mTAL cells. Additional studies showed that TNF produced by mTAL cells inhibits ??Rb uptake, an in vitro correlate of natriuresis, in an autocrine- and COX-2-dependent manner. The molecular mechanism for these effects likely includes inhibition of Na?-K?-2Cl? cotransporter (NKCC2) expression and trafficking.