Monocyte apoptosis in dialysis patients is Fas ligand-mediated.

BACKGROUND: The mononuclear phagocyte system plays an important role in host defense. Since dialysis patients have been reported to show enhanced leukocytes apoptosis, we evaluated the mechanism of increased apoptosis of monocytes in dialysis patients. METHODS: Apoptotic studies were carried out on monocytes isolated from dialysis patients as well as healthy subjects. The effect of dialysis sera and membranes was evaluated on monocyte apoptosis as well as monocyte expression of proapoptotic proteins such as Fas and FasL. To confirm the role of FasL, we evaluated the effect of activated secretory products on T cell apoptosis. In addition, we studied FasL content of dialysis sera and supernatants of activated monocytes. RESULTS: Monocytes isolated from dialysis patients (MDP) showed a greater magnitude of apoptosis when compared to monocytes isolated from healthy subjects (MHS) (MHS, 3.6 +/- 1.1% vs. MDP, 24.3 +/-1.4%). Sera of hemodialysis patients (SHD) promoted (p < 0.001) apoptosis of MHS when compared to pooled control sera (HPS) (HPS, 0.8 +/- 0.5% vs. SHD, 11.5 +/- 0.5% apoptotic cells/field). Dialysis membranes, cellulose acetate membranes in particular, promoted monocyte apoptosis. Interestingly, anti-FasL antibodies partly inhibited dialysis sera-induced monocyte apoptosis. Dialysis membranes also modulated monocyte expression of both Fas and FasL. Secretory products of activated monocytes also promoted T cell apoptosis. Dialysis sera and activated monocyte secretory products showed increased FasL content. CONCLUSIONS: These results suggest that dialysis patients have an increased rate of monocyte apoptosis, which is mediated through a uremic milieu (serum factors). One of these serum factors seems to be FasL. In addition, dialysis membranes seem to promote apoptosis independent of the uremic milieu. The present study provides a mechanistical insight into the enhanced apoptosis of monocytes in dialysis patients.

Fas-mediated apoptosis of neutrophils in sera of patients with infection.

In the presence of infection, neutropenia is considered to be a marker of poor prognosis; conversely, neutrophilia may not be a determinant of a better prognosis. Since apoptotic neutrophils are compromised functionally, we evaluated the effect of infection on neutrophil apoptosis. The rate of apoptosis was greater for neutrophils isolated from patients with infection than for healthy controls. Escherichia coli did not directly modulate the rate of neutrophil apoptosis. However, sera from infected patients promoted (P < 0.001) neutrophil apoptosis. Interestingly, the sera of patients with different types of infection (gram negative, gram positive, or culture negative) exerted a more or less identical response on neutrophil apoptosis. Sera of infected patients showed a fivefold greater content of FasL compared to controls. Moreover, anti-FasL antibody partly attenuated the infected-serum-induced neutrophil apoptosis. In in vitro studies, E. coli enhanced monocyte FasL expression. Moreover, conditioned media prepared from activated macrophages from control mice showed enhanced apoptosis of human as well as mouse neutrophils. On the contrary, conditioned media prepared from activated macrophages isolated from FasL-deficient mice induced only a mild degree of neutrophil apoptosis. These results suggest that neutrophils in patients with infection undergo apoptosis at an accelerated rate. Infection not only promoted monocyte expression of FasL but also increased FasL content of the serum. Because the functional status of apoptotic cells is compromised, a significant number of neutrophils may not be participating in the body's defense. Since neutrophils play the most important role in innate immunity, their compromised status in the presence of infection may transfer the host defense burden from an innate response to acquired immunity. The present study provides some insight into the lack of correlation between neutrophilia and the outcome of infection.

Role of 14-3-3epsilon, c-Myc/Max, and Akt phosphorylation in HIV-1 gp 120-induced mesangial cell proliferation.

Focal glomerulosclerosis (FGS) is the predominant glomerular lesion in patients with human immunodeficiency virus (HIV)-associated nephropathy. Initial mesangial cell hyperplasia and subsequent hypoplasia are common features of FGS. In the present study we evaluated the effect of HIV-1 glycoprotein (gp) 120 on human mesangial cell (HMC) growth. HIV-1 gp 120 stimulated HMC proliferation at lower concentrations, whereas it suppressed cell proliferation at higher concentrations. In parallel to the modulation of cell growth, gp 120 at low concentrations resulted in an increase in the expression of c-Myc, Max, and 14-3-3epsilon proteins and phosphorylation of ATP-dependent tyrosine kinases (Akt) at Ser(473). However, the expression of these proteins decreased with increasing concentrations of gp 120. Furthermore, gp 120 also exhibited a dose-dependent inhibition of Akt phosphorylation at Ser-473 without any significant alteration of Akt expression. Little or no effects of gp 120 were observed on the expression of extracellular signal-regulated kinase (ERK), phospho-ERK, Bcl-2, and Bax proteins. At a higher concentration, gp 120 not only promoted HMC apoptosis but also enhanced expression of Fas and FasL. These results suggest that HIV-1 gp 120 induces alterations in conflicting survival signaling pathways that contribute to the potential dual effects of gp 120 in promoting or inhibiting HMC proliferation.

Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats.

Kidney aging has been recognized as a chronic process of compromised renal function and structural changes in the tubulointerstitium and glomerulus. Cell senescence is associated with alterations in cell structure and function, including expression of cytokines and structural and regulatory components of extracellular matrix proteins. In this investigation, we tested the hypothesis that senescent renal cells may accumulate in vivo with advancing age. We also evaluated the expression of transforming growth factor (TGF)-beta1 and p21WAF1/CIP1 in aging kidneys. Sprague-Dawley rats at the ages of 3, 12, and 24 months were used for this study. Renal tissues were processed for morphometric and senescence analysis. Expression of TGF-beta1 and p21WAF1/CIP1 was evaluated by Northern or Western blot analysis and immunohistochemistry. Substantial tubulointerstitial injury occurred at the age of 12 months, but significant glomerular structure alteration was observed at the age of 24 months. Tubular cells developed senescence, which was detected by beta-galactosidase staining. This staining increased in frequency and intensity with age. Renal cortices showed a significant increase in the mRNA expression for TGF-beta1 and protein level for p21WAF1/CIP1. The enhanced expression of TGF-beta1 and p21WAF1/CIP1 was localized in the tubulointersititial cells. These data suggest that tubular cells undergo senescence and express increased TGF-beta1 and p21WAF1/CIP1 with advancing age. These age-related cellular and molecular alterations may play an important role in the initiation and/or progression of tubulointerstitial fibrosis and glomerulosclerosis in aging.

Morphine stimulates mesangial cell TNF-alpha and nitrite production.

BACKGROUND: Intravenous opiate abusers are susceptible to develop heroin and HIV-associated nephropathies; however, the role of opiates in the development of these kidney lesions is not clear. Patients with opiate addiction are prone to recurrent infections. METHODS: The effect of morphine was studied on the generation of TNF-alpha with or without LPS (lipopolysaccharide) by cultured mouse mesangial cells. In addition, the effect of morphine was evaluated on mesangial cell nitrite production. To evaluate the role of opiate receptors, we studied the effect of naloxone and naltrexone on mesangial cell TNF-alpha and nitrite production. To determine the role of TNF-alpha on mesangial cell nitrite production, we examined the effect of anti-TNF-alpha antibody on morphine-induced nitrite production. Assay of TNF-alpha and nitrite production was carried by ELISA and Griess method respectively. RESULTS: Morphine alone did not enhance the generation of TNF-alpha by mesangial cells, however, an enhanced (P < 0.001) TNF-alpha production was observed when mesangial cells were first treated with morphine for 18 h and then activated further with LPS. Maximum release of TNF-alpha was seen at a concentration of 10(-12) M of morphine. Opiate receptor antagonists (naloxone and naltrexone) inhibited the effect of morphine. Morphine also amplified (P < 0.0002) the effect of LPS on mesangial cell nitrite production. Anti-TNF-alpha antibody attenuated morphine induced nitrite generation. CONCLUSION: We conclude that morphine stimulates the generation of TNF-infinity by LPS-activated mesangial cells. This effect of morphine seems to be opiate receptor mediated and has a downstream effect in the form of mesangial cell nitrite generation. The present in vitro study provides the basis for a hypothesis that morphine may be playing a role in the development of heroin and HIV-associated nephropathies.

Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta.

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta antibody inhibited (P < 0.001) the morphine-induced apoptosis in J774 cells (control 0.7 +/- 0.4%; 10-6 M morphine 23.5 +/- 0.7%; anti-TGF-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta by morphine-treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine-induced bax expression was inhibited by anti-TGF-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.

Human glomerular epithelial cell express CD4 and interaction with gp120 protein promotes PYK2 tyrosine phosphorylation.

Focal segmental glomerulosclerosis (FSGS) is the predominant glomerular lesion in patients with HIV infection. Visceral glomerular epithelial cell (vGEC) injury is a key feature of this glomerular lesion. However, the exact mechanism of HIV-1-induced vGEC injury is not clear. We studied the presence of CD4 (HIV-1 receptor) in vGECs. vGECs were cultured from human kidneys and used during the 5th to 10th passages. Immunocytochemical studies were carried out to visualize CD4 receptors in these cells. Protein and RNA were extracted from vGECs and renal cortical tissues. Western and Northern blots were generated and probed for the expression of CD4. To determine the downstream effect of ligand receptor interaction, vGECs were treated either with variable concentrations of HIV-1 gp120 protein (0.001 to 0.1 microg/ml) for 1 min or with a fixed dose of gp120 protein (0.01 microg/ml) for variable time periods (0 to 10 min), and at the end of the incubation period, tyrosine phosphorylation of pyk2 was studied. Immunocytochemical studies showed the presence of CD4 receptors in vGECs. Western and Northern blot studies confirmed the presence of CD4 expression in these cells. gp120 protein promoted vGEC tyrosine phosphorylation of pyk2 in a dose- and time-dependent manner. The present study provides a mechanistical insight for the role of HIV-1 in the development of glomerular injury in patients with HIV infection.

Morphine promotes apoptosis in Jurkat cells.

Patients with intravenous heroin addiction are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose-dependent manner. DAGO, a specific mu receptor agonist, also promoted Jurkat cell apoptosis. DNA isolated from morphine-treated Jurkat cells and T lymphocytes also showed integer multiples of 200 base pairs. Superoxide dismutase (SOD) enhanced lymphocyte apoptosis; whereas catalase attenuated the morphine-induced apoptosis of Jurkat cells as well as of T lymphocytes. Morphine-treated Jurkat cells also showed a decreased expression of bcl-2 and an enhanced expression of bax. In addition, morphine-treated Jurkat cells showed activation of caspase-3. These results indicate that morphine-induced T lymphocyte apoptosis may be mediated through the generation of reactive oxygen species. The change in ratio of bax and bcl-2 seems to tilt the balance toward apoptosis, leading to the activation of caspase-3. This study provides further support for the hypothesis that morphine may be directly compromising immune function by enhancing apoptosis of T lymphocytes in patients with heroin addiction.

Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3.

Aging is associated with altered immune function. We previously reported that splenocytes and thymocytes undergo apoptosis with aging in rats. In the present study, we examined the expression of genes associated with apoptosis in splenocytes and thymus in aging rats. We evaluated the expression of bax, interleukin 1-beta-converting enzyme (ICE)/ced-3 protease family, caspase-3 and tumor suppressor gene p53. Rats in age groups of 6, 24, 48, and 96 weeks were sacrificed; thymocytes and splenocytes were isolated followed by lysis in a modified RIPA buffer containing protease inhibitors. Western blot analysis of proteins was performed by probing immunoblots with antibodies against p53, bax and PARP (poly ADP-ribose polymerase). Increased aging was associated with enhanced expression of bax, p53 and cleavage of PARP by Caspase-3. The expression of p53 and cleavage of PARP indicates the presence of damaged DNA; nevertheless, the cleavage of PARP or activation of caspase-3 may be playing an important role in the initiation of early events in apoptosis. These results suggest that aging of splenocytes and thymocytes is associated with the expression of cell death genes. The present study provides an insight into age-associated altered immune function.

Morphine enhances macrophage apoptosis.

Laboratory data indicate that morphine decreases the numbier of peritoneal and alveolar macrophages (Mphi) and compromises their phagocytic capability for immune complexes and bacteria. We hypothesize that morphine decreases the number of, as well as compromises the phagocytic capability of, Mphi by programming their death. We studied the effect of morphine on Mphi apoptosis in vivo as well as in vitro. Peritoneal Mphi harvested from morphine-treated rats showed DNA fragmentation. Morphine enhanced murine Mphi (J 774.16) apoptosis in a dose-dependent manner. Human monocytes treated with morphine showed a classic ladder pattern in gel electrophoretic and end-labeling studies. Morphine promoted nitric oxide (NO) production both under basal and LPS-activated states. N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine monoacetate (L-NMMA), inhibitors of NO synthase, attenuated the morphine-induced generation of NO by Mphi. Morphine also enhanced Mphi mRNA expression of inducible NO synthase (iNOS). Since morphine-induced Mphi apoptosis was inhibited by L-NAME and L-NMMA, it appears that morphine-induced Mphi apoptosis may be mediated through the generation of NO. Morphine promoted the synthesis of Bax and p53 proteins by Mphi. Moreover, IL-converting enzyme (ICE)-1 inhibitor attenuated morphine-induced Mphi apoptosis. These studies suggest that morphine activates the induction phase of the apoptotic pathway through accumulation of p53. The effector phase of morphine-induced apoptosis appears to proceed through the accumulation of Bax and activation of ICE-1. The present study provides a basis for a hypothesis that morphine may be directly compromising immune function by promoting Mphi apoptosis in patients with opiate addiction.

Differential expression and autoradiographic localization of atrial natriuretic peptide receptor in spontaneously hypertensive and normotensive rat testes: diminution of testosterone in hypertension.

Previous studies have shown that the diuretic hormone atrial natriuretic peptide (ANP) also regulates the steroidogenic responsiveness in isolated Leydig cells from mouse and rat testes. In the present study, we examined the distribution of specific receptors for ANP and C-type natriuretic peptide (CNP) in the testicular compartments of 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). We used an in vitro autoradiographic procedure on slide-mounted frozen testicular sections to localize the receptors of the natriuretic peptide hormone family using 125I-ANP and 125I-CNP as radioligands. A high level of specific 125I-ANP binding sites was localized largely in the Leydig cells of the interstitial compartment; other testicular cells were not significantly labeled. On the other hand, no significant difference was observed in 125I-CNP binding sites in the testicular cells of SHR and WKY. Semiquantitative analysis of the binding sites indicated that the density of 125I-ANP receptor binding in Leydig cells of WKY testis was ninefold higher than in those of SHR testis. A moderate level of 125I-ANP binding was also observed in seminiferous tubules, particularly in the spermatids of both SHR and WKY. 125I-ANP binding in WKY spermatids was approximately 2.5-fold higher than in SHR spermatids. Northern blot analysis showed that mRNA specific for guanylyl cyclase type A (Npra) was expressed at approximately twofold higher levels in WKY than in SHR testis. ANP (1 x 10(-8) mol/L) stimulated fourfold to fivefold increased levels of testosterone production in isolated Leydig cells from normotensive WKY compared with those from SHR. These findings support a new physiological role of ANP in Leydig cells, in which a functional relationship seems to exist between testicular ANP receptor expression and testosterone production and the state of hypertension in SHR.

Differential distribution of inhibin in the anatomical regions of monkey stomach.

Inhibin, a 10.7 kD FSH (follicle-stimulating hormone) suppressing prostatic peptide has been shown to be synthesized and localized in stomach specimen of monkey. In vitro incorporation of labelled amino acid (3H-leucine) into inhibin followed by specific immunoprecipitation by antiserum to inhibin demonstrated an in vitro de novo biosynthesis of inhibin by monkey stomach. Moreover, the synthesis of inhibin was found to be maximum in fundic zone of gastric mucosa compared to cardiac and antral zone. This was supported by immunohistochemical study of three anatomically different regions, especially wherein fundic zone showed intense positive staining for inhibin. Furthermore, the above data was supplemented by quantitative study of tissue inhibin content by RIA which revealed that the fundic zone of gastric mucosa has a much higher concentration of inhibin than cardiac and antral region. The relationship of zonal concentration of inhibin to gastric anatomy appears to be a noteworthy observation and may serve as an useful tool in our understanding of gastric metabolism and activity.

De novo biosynthesis and localization of immunoreactive inhibin (10.7 kDa) in normal human stomach.

We have previously reported the occurrence of inhibin-like peptide in gastric juice of normal men. In the present investigation, normal gastric mucosa was shown to synthesize inhibin, in vitro, as measured by 3H-leucine incorporation (Maximum at 18 h). Furthermore, the immunohistochemical localization studies demonstrated its presence in the acid secreting parietal cells and basal region of foveolar epithelium of gastric mucosa. Surprisingly, the protein secreting zymogen cells remained unstained.

Possible involvement of the accessory sex glands in the regulation of pituitary hormones.

A time-course alteration in the blood levels of gonadotrophin was evaluated in male rats aged 49 days after: (a) removal of the seminal vesicles, (b) bilateral orchidectomy, or (c) bilateral orchidectomy together with removal of the seminal vesicles. Age-matched sham-operated animals served as controls. Removal of the seminal vesicles resulted in elevation in the blood levels of LH, FSH and prolactin compared to controls. As expected, bilateral orchidectomy also resulted in the elevation of serum gonadotrophins. In the absence of both the seminal vesicles and the testes, LH and FSH levels were increased further compared to orchidectomy alone. Thus the present study suggests the presence of a feedback mechanism between the seminal vesicles and pituitary which is more pronounced in the absence of the testes.