Nora Gibbons

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 p 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. J. Leukoc. Biol. 66: 650–658; 1999.

Prevalence and predictors of rhabdomyolysis in patients with hypophosphatemia

PURPOSE We undertook this study to determine the prevalence and predictors of rhabdomyolysis in the hypophosphatemic state. PATIENTS AND METHODS To identify patients with hypophosphatemia, we reviewed medical admissions for the period of January through December 1989. The hypophosphatemic state was considered whenever the serum phosphate was less than or equal to 2.0 mg/dL. Rhabdomyolysis secondary to hypophosphatemia was defined when serum creatine kinase levels were greater than or equal to 224 IU/L; it occurred within 72 hours of the hypophosphatemic episode; and it subsequently normalized. Patients who had any other independent etiology for rhabdomyolysis were excluded. Clinical and biochemical characteristics of patients with rhabdomyolysis (Group I) and patients without rhabdomyolysis (Group II) were compared. Variables that predicted rhabdomyolysis in hypophosphatemia were identified by stepwise logistic regression using a backward elimination procedure. RESULTS One hundred twenty-nine patients were found to have hypophosphatemia. Forty-six (Group I) of 129 patients (36%) showed biochemical evidence of rhabdomyolysis. There was no difference in serum phosphate and potassium concentrations between Group I and Group II patients. Patients in Group I showed higher values for serum osmolality (p less than 0.05), serum glutamic oxaloacetic transaminase (p less than 0.001), chloride (p less than 0.01), and blood urea nitrogen (less than 0.05) when compared with Group II patients. When biochemical profiles of patients with rhabdomyolysis were evaluated on the day of their peak creatine kinase level, only 16 patients were hypophosphatemic, and the majority of patients showed a transient increase in serum phosphate levels because of ongoing muscle cell injury. Of 17 potential predictors, six variables emerged including sodium, chloride, glucose, blood urea nitrogen, uric acid, and osmolality. These variables provided high sensitivity (0.88) as well as moderate specificity (0.79) for predicting the occurrence of rhabdomyolysis in hypophosphatemia. CONCLUSION We conclude that rhabdomyolysis commonly occurs in the hypophosphatemic state and that at times severe hypophosphatemia as an etiology may be masked because of ongoing rhabdomyolysis. Serum sodium, chloride, glucose, blood urea nitrogen, uric acid, and osmolality have a predictive role for the occurrence of rhabdomyolysis in the hypophosphatemic state that shows a high specificity and a moderate sensitivity.

Morphine Induces Splenocyte Apoptosis and Enhanced mRNA Expression of Cathepsin-B

Morphine has been demonstrated to modulate immune function. We studied whether morphine modulates apoptosis of splenocytes. Splenocytes were isolated from control and morphine treated rats. Splenocytes isolated from morphine treated rats showed increased percentage (P < 0.001) of apoptosis when compared to splenocytes isolated from untreated rats (control, 4.7 ± 1.0% apoptotic splenocytes/field vs. morphine, 47.8 ± 3.4% apoptotic splenocytes/field). These results were further confirmed by gel electrophoresis as well as by end-labeling DNA of splenocytes isolated from control and morphine treated rats. Splenocytes from morphine treated rats showed a classical ladder pattern with integer multiples of 180 base pairs. Splenocytes from morphine treated rats also showed increased mRNA expression of cathepsin-B, a gene associated with active cell death. These results suggest that morphine may also be modulating immune function by enhancing apoptosis of splenocytes.

Effect of morphine on renomedullary interstitial cell proliferation and matrix accumulation.

Renal interstitial scarring is an important feature of heroin-associated nephropathy. We studied the effect of morphine, an active metabolite of heroin, on cultured rat renal medullary interstitial cell (RMIC) proliferation and matrix accumulation. Morphine (10(-12) M) enhanced (p < 0.001) the proliferation of RMIC (control, 15.0+/-0.5 vs. morphine, 20.4+/-1.1 x 10(4) cells/ml). This effect of morphine was dose and time dependent. [3H]thymidine and bromodeoxyuridine incorporation studies confirmed the mitogenic effect of morphine on RMIC. Morphine also enhanced mRNA expression for c-jun and c-myc on RMIC. However, nalbuphine, a non-addicting alkaloid did not modulate the proliferation of RMIC. Morphine enhanced the accumulation of collagen type I in a dose-dependent manner and also increased (p < 0.001) the accumulation of collagen type III at a high concentration (control, 1,291+/-55.8 vs. morphine, 10(-4) M, 2,697.6+/-257.8 ng/microg protein). Morphine did not modulate the accumulation of laminin or fibronectin. Neutralizing antibody to IL-6 inhibited the effect of morphine on RMIC. H7, a protein kinase C inhibitor, also attenuated the morphine-induced RMIC proliferation. The present study provides a basis for a hypothesis that morphine may be playing a role in the development of renal interstitial pathology in patients with heroin addiction.

Fas-Mediated Apoptosis of Neutrophils in Sera of Patients with Infection

ABSTRACT 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 bodys 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.

Hyperlipoproteinemia in spontaneously diabetic guinea pigs

A colony of Hartley guinea pigs that exhibit hyperglycemia, glucosuria, and hypertriglyceridemia characteristic of human diabetes mellitus was developed. Initially, a group of guinea pigs that had normal serum glucose concentrations (less than or equal to 200 mg/dL of serum) at 3 to 4 weeks of age was obtained; however, in some of the animals progressively severe hyperglycemia (300 to 500 mg/dL of serum) and glucosuria (greater than 2 g of glucose/24 h) occurred as the animals matured. In addition, the animals exhibiting hyperglycemia and glucosuria had plasma insulin concentrations that were similar to those animals that were not hyperglycemic. The diabetic animals were found to be hypertriglyceridemic, with plasma triglyceride levels of 140 to 290 mg/dL at four months of age. Nondiabetic animals (plasma glucose concentration of less than or equal to 200 mg/dL and no glucosuria) had plasma triglyceride concentrations between 37 and 76 mg/dL. Lipoprotein analysis of plasma from nondiabetic and diabetic animals indicated that the diabetics had a fourfold increase in VLDL triglyceride and protein concentrations. The VLDL had an abnormal apolipoprotein composition and had reduced levels of apoprotein-E. The progeny from the mating of diabetic males and females also exhibited the diabetic trait, suggesting that the origin of the disease is genetic. This colony of guinea pigs is being further investigated as a suitable model for the study of the hyperlipoproteinemia of human noninsulin-dependent diabetes mellitus.

Escherichia coli-Macrophage Interactions Modulate Mesangial Cell Proliferation and Matrix Synthesis

Patients with chronic renal interstitital diseases often develop glomerular lesions (focal segmental glomerular sclerosis). Because mesangial expansion (enhanced mesangial cell (MC) growth and matrix accumulation) has been demonstrated to precede the development of focal segmental glomerulosclerosis, we studied the effect of the interaction between bacteria such as Escherichia coli and macrophages on MC proliferation and matrix synthesis. We determined the effect of control media (CM), E. coli supernatant (ESP), serum-free macrophage supernatant (MSP), and E. coli-treated macrophage supernatants (HB101-MSP, H10-MSP) on the proliferation of MCs and synthesis of laminin (a component of mesangial matrix). ESP did not alter MC growth, whereas E. coli MSP increased the mean MC number by 5- to 6-fold when compared to cells treated with CM. Both HB101-MSP and H10-MSP stimulated greater (p < 0.05) incorporation of [3H]thymidine when compared with MSP (HB101-MSP 3.1 +/- 0.4, H10-MSP 2.7 +/- 0.3 vs. MSP 1.6 +/- 0.2 x 10(6) cpm/micrograms protein). When MC proliferation was judged by incorporation of bromodeoxyuridine, both HB101-MSP- and H10-MSP-treated cells showed a greater (p < 0.01) number of proliferating cells compared with cells treated with either MSP or CM. MC treated with H10-MSP grew in a specific pattern and showed a tendency to form hillocks (foci of cell proliferation and matrix aggregation). Both HB101-MSP and H10-MSP enhanced (p < 0.01) synthesis of laminin compared with CM. HB101-MSP-induced enhanced laminin synthesis was attenuated when MCs were treated with anti-transforming growth factor (TGF)-beta antibodies. HB101-MSP also increased mRNA expression of TGF-beta by MCs. These results indicate that E. coli-macrophage interaction has the potential to cause mesangial expansion.

Morphine Enhances Deposition of Ferritin-Antiferritin Complexes in the Glomerular Mesangium

Since increased mesangial accumulation of matrix has been considered to be an important event in the development of focal glomerulosclerosis, we investigated whether morphine, an active metabolite of heroin, can modulate mesangial accumulation of immune complexes. Control or morphine-dependent rats were administered intraperitoneal ferritin (8 mg/100 g body weight) daily for 6 weeks. Body weight, blood pressure, serum creatinine, 24-hour urinary protein and creatinine excretion rates were measured at 3-week intervals. Rats were sacrificed at the end of 6 weeks and kidney tissue was studied by light, immunofluorescence and electron microscopy. Serum creatinine levels and urinary protein excretion rates were not different between control and morphine-dependent rats. All morphine-dependent rats developed hematuria, whereas only 1 control rat developed hematuria. Light microscopy revealed no proliferation of mesangial cells and only a minimal increase in the mesangial matrix. Electron-microscopic studies showed deposition of immune complexes in the mesangial region. Mesangial cells showed aggregation of ferritin in lysosomes. Immunofluorescence studies revealed the presence of IgG staining predominantly in the mesangial region. The majority (60%) of morphine-dependent rats showed a diffuse mesangial deposition of IgG when compared to control rats (83%) who showed only focal deposition. These results indicate that morphine enhances deposition of immune complexes in the mesangium. Morphine-induced matrix but may also change its quality. This may play a pathogenic role in the development of glomerular lesions in patients who abuse opiates.

Dexamethasone attenuates oxidation of extracellular matrix proteins by human monocytes.

In response to infection or in immune complex-mediated diseases, inflammatory cells may oxidatively damage extracellular matrix (ECM) proteins. In this study we evaluated whether human monocytes could oxidize ECM and whether this could be modulated by exposure to LPS, IgG complexes, and dexamethasone (DEX). Wells in tissue culture plates were coated with the ECM preparation Matrigel. Porous inserts with or without the human monocyte cell line THP-1 were placed into ECM-containing wells and cells were exposed to control conditions or to LPS (10 ng/ml), IgG complexes (200 and 500 microg/ml), or DEX (10(-7) and 10(-6) M). ECM was then subjected to Western blot analysis using an antibody to oxidized protein. In addition, Western blot analysis was carried out on DEX-treated cells to evaluate expression of the NADPH oxidase components p67-phox and gp91-phox. THP-1 cells enhanced ECM oxidation and this effect was augmented by LPS and by IgG aggregates. Preincubation of cells with DEX attenuated ECM oxidation and was also associated with decreased expression of p67-phox and gp91-phox. These findings suggest that human monocytes can oxidize ECM proteins and that this may be modulated by IgG complexes and LPS. Dexamethasone appears to attenuate ECM oxidation and a better understanding of this mechanism might allow for interventions to minimize oxidative damage to ECM proteins by monocytes in infectious and inflammatory states.

Opioids Modulate Migration, Spreading and Adherence of Mesangial Cells

Glomerular mesangial cells are considered to be modified smooth muscle cells and seem to play a role in the maintenance of glomerular hemodynamics. The present study was carried out to determine the effect of opioids on adhesiveness, spreading and migration of mesangial cells. Morphine enhanced spreading of mesangial cells at early time periods (5 mini, control 8 +/- 2% vs. morphine 15 +/- 1%, p < 0.05; 15 min, control 21 +/- 5% vs. morphine 38 +/- 2%, p < 0.05) as well as at later time periods when compared to control cells (at 2 h, control 23 +/- 1% vs. morphine 49 +/- 1%, p < 0.001; at 3 h, control 28 +/- 3% vs. morphine 63 +/- 2%, p < 0.05). beta-Endorphin also enhanced (p < 0.001) spreading of mesangial cells (at 2 h, control 23 +/- 1% vs. beta-endorphin 48 +/- 3%; at 3 h, control 28 +/- 3% vs. beta-endorphin 65 +/- 1%). Morphine decreased adhesion of mesangial cells to the plastic substrate at 24 h as well at 48 h when compared to the control cells. Naloxone attenuated the effect of morphine on adhesion to the substrate. Morphine enhanced (p < 0.05) migration (percentage of denuded area covered by mesangial cells when compared to control cells (control 26.07 +/- 1.08% vs. morphine 37.5 +/- 2.94%; n = 9). Since the morphine-induced decreased adhesiveness could be attenuated by naloxone, this effect of morphine on mesangial cells appears to be mediated by opioid receptors.