Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

INTRODUCTION: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. METHODS: Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. RESULTS: ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). DISCUSSION: Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. CONCLUSION: Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.

OS053. Trophoblast invasion in the mesometrial triangle of atp infused pregnant rats.

INTRODUCTION: Poor placentation (disturbed and decreased trophoblast invasion) is a hallmark of preeclampsia (PE), which is a major complication of pregnancy. Unfortunately, the cause and mechanism of disturbed trophoblast invasion is still unknown. OBJECTIVES: The pro-inflammatory agent ATP has been shown to induce PE-like signs, after a single infusion in pregnant rats. These PE-like characteristics include proteinuria and decreased fetal weight. Since purinergic ATP receptors are expressed on trophoblast cells, we aimed to study the effect of ATP infusion on trophoblast invasion in pregnant rats in this pilot study. METHODS: Pregnant rats received a single ATP (n=4) or saline (control,ni=5) infusion via a permanent jugular vein cannula on day 14 of pregnancy. At the time of maximal trophoblast invasion (day 17 of pregnancy) rats were sacrificed and placentas with mesometrial triangle were collected, fixed in zinc-buffer and embedded in paraffin. 4 µm sections were stained with monoclonal α-cytokeratin antibodies. In the mesometrial triangle, the maternal part of the rat placenta, the percentage of surface area of trophoblast invasion was evaluated using computerized image analysis. Also, the depth and width of invasion were analyzed by subdividing the mesometrial triangle in three concentric depth levels of equal width. In addition, trophoblast invaded versus non-invaded spiral arteries in the mesometrial triangle were quantified. RESULTS: In the mesometrial triangle, no changes in percentage of surface area of trophoblast invasion and percentage of invaded spiral arteries were observed after ATP infusion. However, the pattern of trophoblast invasion appeared to be disturbed in ATP infused rats, with a decreased depth of invasion and an increased width of invasion, resulting in a trend towards a decreased depth/width ratio of trophoblast invasion in ATP infused rats. CONCLUSION: In this (pilot) study we showed an altered trophoblast invasion pattern in the mesometrial triangle of the placenta, although no significant differences in the total surface area of trophoblast invasion were seen in experimental versus control pregnant animals. e mechanism by which ATP induces this altered trophoblast invasion pattern and its potential contribution to the pathophysiology of this experimental PE in the pregnant rat awaits further investigation.

OS056. Angiotensin II sensitivity and endothelial dysfunction afterexperimental preeclampsia.

INTRODUCTION: Women who suffered from preeclampsia (PE) have an increased risk for cardiovascular and renal diseases later in life. Although the exact mechanisms underlying this relationship are unknown, they may relate to an increased sensitivity to angiotensin II (Ang-II) and endothelial dysfunction during a preeclamptic pregnancy, which may persist after PE. Recently, we showed vascular hypersensitivity to Ang-II and disturbed endothelial cell function in experimental PE in rats as compared to healthy pregnant rats. OBJECTIVES: To study whether vascular hypersensitivity to Ang-II and endothelial dysfunction persist postpartum in experimental PE. METHODS: In this ongoing study, we thus far included non-pregnant rats (NP;n=9), formerly healthy pregnant rats (HP;n=9) and formerly experimental preeclamptic rats (PE; infusion of a low dose endotoxin; n=16). Six weeks after pregnancy, animals from each group were treated with Ang-II (osmotic minipump; 200ng/kg/min;NP: n=5;HP: n=6;PE: n=8) or were sham treated (NP: n=4;HP: n=3;PE: n=8). Blood pressure was measured in all rats one day before and weekly after Ang-II or sham treatment (for three weeks). At termination, the aortas of sham operated rats were obtained. Aortic rings (2mm) were mounted for isotonic measurement of vasotonus. Endothelium-dependent acetylcholine- (ACh) mediated vasodilation was studied in phenylephrine-preconstricted rings in the presence of vehicle, N(G)-nitro-L-arginine methyl ester, indomethacin or both, followed by full concentration response curves for ACh (10(-8)M-10(-4)M). Ang-II sensitivity was assessed by obtaining full concentration response curves (10(-10)M-10(-6)M). AT-1 and AT-2 receptor sensitivity was determined by administration of Ang-II in the presence of the AT-1 receptor blocker losartan, or the AT-2 receptor blocker PD123319. RESULTS: Our results indicate no difference in mean (±SD) systolic blood pressure (SBP) between the three groups six weeks after delivery (NP: 129(±7);HP: 127(±9);PE: 123(±10)mmHg;p=0.248). However, after three weeks of Ang-II treatment, a trend was found towards a stronger increase in SBP in PE rats as compared to HP rats (45.7(±18.9)% vs 63.4(±20.1)% respectively;p=0.081). Although we found no differences in in-vitro Ang-II sensitivity between the three groups, NP rats showed a trend towards an increased sensitivity of the AT-2 receptor to Ang-II compared to both groups of formerly pregnant rats. Total ACh-mediated endothelial relaxation was not different between the three groups. However, contribution of both NO and EDHF components to ACh-mediated relaxation seemed decreased in both groups of formerly pregnant rats as compared to the NP rats. CONCLUSION: These preliminary data suggest that healthy rats that suffered from preeclampsia during pregnancy have increased in-vivo sensitivity to Ang-II postpartum as compared to rats with an uncomplicated pregnancy. Whether these differences are related to in-vitro- changes in Ang-II sensitivity or changes in endothelial function remains to be established.

Complement dependency of splenic localization of pneumococcal polysaccharide and conjugate vaccines.

The immune response to polysaccharides is initiated when polysaccharides bind complement factor C3d, and these polysaccharide-C3d complexes subsequently localize on splenic marginal zone B cells strongly expressing CD21 (complement receptor 2). Infants and children under the age of 2 years have low or absent expression of CD21 on their marginal zone B cells, and consequently do not adequately respond to polysaccharides. In contrast, polysaccharide-protein conjugate vaccines are able to induce antibodies at this young age. Conjugate vaccines apparently overcome the necessity for CD21-C3d interaction for an antipolysaccharide immune response. We demonstrate in a rat model that localization of pneumococcal polysaccharides on splenic marginal zone B cells indeed is complement dependent. We also show that pneumococcal conjugates do not specifically localize on splenic marginal zone B cells and that splenic localization of polysaccharide conjugates is independent of the presence of complement. Thus, the induction of antipolysaccharide antibodies by conjugate vaccines apparently can occur independently of CD21-C3d interaction. These basic findings may explain the effectiveness of conjugated vaccines in young children and may open the way for their application in other patient groups.

Effects of multidose combination chemotherapy on the humoral immune system.

Patients receiving multidose combination chemotherapy are at risk for severe, life-threatening infections, caused by among others encapsulated bacteria like Streptococcus pneumoniae. The splenic marginal zone is essential in the initiation of immune responses to S. pneumoniae. We analyzed effects of multidose combination chemotherapy on B-cell subpopulations. Immune response capacity was evaluated by using Pneumovax (PPS) or Tetavax (TT) as antigenic challenge. Three days after finishing therapy, all B-cell subpopulations in bone marrow and spleen were severely reduced, including the mature marginal zone B-cell population. When analyzing the anti-PPS immune response capacity at 3 days after finishing therapy, we found that the IgM antibody levels did not differ significantly from control immunized rats. The IgG antibody levels were significantly lower compared to control immunized rats but still significantly higher compared to unimmunized rats. The depletion of marginal zone B cells by multidose combination chemotherapy most likely contributes to the prolonged period that patients are at risk for developing severe infections after chemotherapy, despite the capacity to generate sufficient antibody levels. It is conceivable that the local (temporary) loss of immunological memory, together with the supposed inability to generate a humoral response in a short time frame, plays an important role in this vulnerability.

After chemotherapy, functional humoral response capacity is restored before complete restoration of lymphoid compartments.

Chemotherapy has, besides the beneficial effects, several adverse effects. Suppression of the immune system is one of the most important problems. Infections caused by encapsulated bacteria like Streptococcus pneumoniae are responsible for a major part of infectious problems during and after treatment. The splenic marginal zone is essential in the initiation of an immune response to encapsulated bacteria. In this study, we analysed the effects of three different cytostatic agents on humoral immune responses. We found a reduced, but detectable immune response capacity at two days after treatment although the marginal zone B cell population is severely reduced at this time point. Twenty-four days after cessation of treatment, the immune response capacity was largely restored although lymphoid compartments were still not completely restored at that time point. Apparently, the presence of only few marginal zone B cells is sufficient to evoke a rise in antibody titres and although antibody titre increases are low, even small rises are most likely clinically relevant.

Nitric oxide inhibition enhances platelet aggregation in experimental anti-Thy-1 nephritis.

In the present paper we studied the role of nitric oxide radicals (NO) on platelet aggregation, fibrinogen deposition, superoxide formation, peroxynitrite formation, hemodynamics, and leukocyte migration in the Thy-1 model of glomerulonephritis. To first study the baseline kinetics of these parameters, groups of anti-Thy-1-treated rats were sacrificed at 1 h, 4 h, 24 h, 3 days, 7 days, and 14 days and compared to controls. Urinary protein excretion was significantly elevated in Thy-1 nephritis at 3 and 7 days. Glomerular macrophages, PMNs, and superoxide anion-positive cells were significantly increased in Thy-1 nephritis. Nitrotyrosine immunoreactivity was absent during the entire study period. Glomerular platelet aggregation was significantly increased in anti-Thy-1 injected rats at 1 h, 4 h, 24 h, and 3 days. Glomerular fibrinogen deposition was significantly elevated at all time points. To elucidate the role of NO in this process, additional groups of anti-Thy-1-injected rats were treated with the NOS inhibitor l-NAME and studied at 24 h. Urinary protein excretion was significantly higher in l-NAME treated Thy-1 rats compared to nontreated Thy-1 rats. Plasma and urine nitrite/nitrate levels were significantly lower in l-NAME-treated Thy-1 rats compared to nontreated Thy-1 rats. Compared to nontreated Thy-1 rats, there were no differences in intraglomerular leukocyte accumulation after treatment with l-NAME. In contrast, we observed a marked increase in platelet aggregation following l-NAME treatment. From these data we conclude that the inflammatory infiltrate in Thy-1 nephritis develops independent of NO radical production, whereas NO radicals prevent the accumulation of platelet aggregates.

Pneumococcal conjugate vaccines overcome splenic dependency of antibody response to pneumococcal polysaccharides.

Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.

Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity.

Although most chemotherapeutic agents are known to cause primarily reduction or suppression of immune responses, surprisingly little is known about the influence of cytostatic agents on lymphoid tissue compartments such as the splenic marginal zone. The marginal zone plays an important role in the defence against encapsulated bacteria, which are potential candidates for postchemotherapeutic infections. We studied the effect of three different cytostatic agents (cisplatin, methotrexate, and cyclophosphamide) on B cell subpopulations in a rat model. Rats received a single dose of a single cytostatic agent and were sacrificed at different time points after treatment. Bone marrow, blood, mesenteric lymph nodes and spleens were analysed by flow-cytometry and immunohistochemistry. All three cytostatic agents showed severe bone marrow depression. CP and MTX showed only mild reduction of cell populations in the spleen. CyPh showed a severe reduction of recirculating follicular B (RF-B) cells and marginal zone B (MZ-B) cells. At day 24 most populations were already recovered, but RF-B cells and MZ-B cells were still reduced. The reduction of the marginal zone and late recovery may imply that, beside the overall increased infection risk due to neutropenia, patients treated with chemotherapy are at risk for developing infections from encapsulated bacteria for a considerable period of time after treatment, extending beyond the period of bone marrow depression.

Chronic blockade of angiotensin II action prevents glomerulosclerosis, but induces graft vasculopathy in experimental kidney transplantation.

Long-term renin-angiotensin system blockade is beneficial in a variety of renal diseases. This study examines the long-term (34 weeks) effects of the angiotensin-converting enzyme inhibitor lisinopril and the angiotensin II receptor type I blocker L158,809 in the Fisher to Lewis rat model of chronic renal transplant failure. Treatment in allografted rats with lisinopril or L158,809 was initiated 10 days after transplantation, or at the time when proteinuria exceeded 50 mg/24 h. Untreated allografts and syngrafts served as controls. In contrast to syngrafts, untreated allografts developed proteinuria, hypercholesterolaemia, interstitial damage, and glomerulosclerosis. Lisinopril or L158,809 treatment in allografts starting at day 10 after transplantation completely prevented this, with the exception of interstitial damage, but this treatment also caused a reduction in blood pressure and renal function. Moreover, the intimal surface area of the renal arteries was dramatically increased in allografts treated with either lisinopril or L158,809 compared with untreated allografted rats. Treatment once proteinuria had developed was less effective in preventing glomerulosclerosis, but also caused less intimal expansion. Thus, chronic renin-angiotensin system blockade preserves glomerular morphology in the absence of proteinuria, but enhances intimal hyperplasia and reduces renal function in experimental transplantation. In view of these results, it should be questioned whether such treatment benefits renal transplant patients in the long term.

Induction of experimental proteinuria in vivo following infusion of human plasma hemopexin.

BACKGROUND: The human plasma constituent hemopexin (Hx), following incubation with renal tissue, is able to induce glomerular alterations in vitro that are similar to those seen in minimal change disease (MCD). Whether this acute phase reactant is also able to induce proteinuria and minimal change-like alterations in vivo is questioned. METHODS: In the first set of experiments, Hx (4.0 mg in 5.0 mL saline) or equal amounts of control fraction, that is, heat-inactivated Hx (HI-Hx), were infused into conscious rats (N = 6) that had been surgically equipped with a cannula inserted into the suprarenal artery (SRA), enabling direct contact of the infusate and the renal microvasculature. Each animal received HI-Hx at day 1 for 15 minutes (flow rate 20.0 mL/h), subsequently followed by saline for seven hours (Flow rate 5.0 mL/h), after which the cannula was disconnected. At day 2, identical infusions in the same rat were carried out, using native Hx. Urine samples collected every 30 minutes during the experiments were monitored for protein content using standard methods. In the second set of experiments, unilateral perfusion was done ex vivo in anesthetized rats with Hx (N = 5) or HI-Hx (N = 3; 1.5 mg/mL; 4.0 mL during 6 min). After reconnection of the circulation, urine samples of both kidneys were collected every 30 minutes during five hours via ureter cannulation. Urinary protein (expressed as the difference in excretion between perfused and nonperfused kidney) was calculated in mg/24 h. In additional experiments, rats were sacrificed two hours after perfusion of Hx or heat-inactivated (control) Hx (first set of experiments) or after five hours (second set of experiments), and kidneys were processed for immunohistochemical and ultrastructural examination. RESULTS: The results of experiment 1 show a significant increase of proteinuria after Hx infusion versus HI-Hx (means +/- SD, 41.91 +/- 16.01 mg/24 h vs. control, 21.22 +/- 5.69 mg/24 h; P

Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues.

Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several antibodies did not react with the expected cell populations in a certain species, or reacted in previously unknown patterns. In addition, different antibodies to the same isoform rarely detected identical cell populations, even within one species. Most of these unexpected immunoreactivities were observed in bronchial epithelial, glomerular epithelial, and vascular smooth muscle cells. These unexpected results usually occurred when the antibodies were tested in other organs or species than that to which they were originally raised. We therefore strongly recommend the use of anti-NOS antibodies only after careful immunohistological and biochemical analysis of their reactivity in the organ and species to be studied.

Expression of iNOS, eNOS, and peroxynitrite-modified proteins in experimental anti-myeloperoxidase associated crescentic glomerulonephritis.

Nitric oxide radicals are recognized as important mediators in various physiological and pathophysiological processes. During inflammation, increased amounts of nitric oxide (NO) are produced, but it is unclear whether NO radicals are either protective or harmful. To obtain more insight into the role of NO in glomerular inflammation, we studied the temporal expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) in conjunction with platelet aggregation, inflammatory cell influx, superoxide anion production cells, and nitrotyrosine formation in an experimental model of anti-myeloperoxidase (MPO) associated necrotizing crescentic glomerulonephritis (NCGN). Brown Norway rats were immunized with MPO in complete Freund's adjuvant (CFA) or CFA alone. After two weeks, the left kidney was perfused with a neutrophil lysosomal extract and H2O2. Rats were sacrificed at 24 hours, four days, and 10 days after perfusion. Kidney sections were stained by immunohistochemistry for eNOS, iNOS, platelets, nitrotyrosines, polymorphonuclear cells (PMN), monocytes, and T-cells. Superoxide anion producing cells were identified by enzyme cytochemistry using diaminobenzidine. Strong staining for eNOS was found in glomerular capillaries and interstitial tubular capillaries and larger vessels from non-perfused kidneys. At 24 hours after perfusion, glomerular and interstitial eNOS staining was greatly reduced, which was associated with massive platelet aggregation. At later time points, eNOS expression was absent in severely damaged glomeruli. Inducible NOS expression was found at all time points in infiltrating inflammatory cells, which by double labeling studies were identified as PMNs and monocytes. The peak in iNOS expression was observed at four days after perfusion but declined thereafter. Superoxide anion and nitrotyrosine generating cells were also found at all time points, but were most abundantly present at four days after perfusion, coinciding with the peak in iNOS expression. Double labeling experiments revealed that most nitrotyrosine generating cells also produced superoxide anions and expressed iNOS. In conclusion, these studies suggest that during the course of anti-MPO associated NCGN, loss of NO production by eNOS in conjunction with NO radical production by iNOS contribute to tissue injury. This is compatible with a protective role for eNOS contrasting with the possibly harmful effects of iNOS in anti-MPO associated NCGN.

Dephosphorylation of endotoxin by alkaline phosphatase in vivo.

Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin is a substance that contains phosphate groups and is usually present in the extracellular space, we studied whether AP is able to dephosphorylate this bacterial product at physiological pH levels. We tested this in intestinal cryostat sections using histochemical methods with endotoxin from Escherichia coli and Salmonella minnesota R595 as substrate. Results show that dephosphorylation of both preparations occurs at pH 7.5 by AP activity. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, we examined the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat. The results show that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus. In view of the endotoxin-dephosphorylating properties of AP demonstrated in vitro, we propose a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role.

Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats.

BACKGROUND & AIMS: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased. NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different NO synthase inhibitors on liver damage and hemodynamics during endotoxemia. METHODS: Rats were injected with lipopolysaccharide (LPS) and received the NOS-inhibitor S-methylisothiourea (SMT) or NG-nitro-L-arginine methyl ester (L-NAME). iNOS induction was assessed by Western blot, immunohistochemistry, and measurement of NO metabolites in plasma and bile. Liver damage was determined by aspartate aminotransferase and alanine aminotransferase and by histology. The effects of both inhibitors on systemic and portal pressure were measured in normal and LPS-treated rats. RESULTS: LPS treatment strongly induced iNOS in inflammatory cells, macrophages, bile duct epithelium, and hepatocytes, especially at the canalicular membrane. LPS-induced liver damage strongly increased after L-NAME. SMT caused a similar reduction of NO production without enhancing liver damage. In LPS-treated rats, SMT increased the systemic and portal pressure significantly more than L-NAME. CONCLUSIONS: During endotoxemia, administration of the NOS-inhibitor L-NAME aggravates liver damage. This liver damage does not seem to be caused by hemodynamic changes. In contrast, SMT caused significant hemodynamic changes but did not increase LPS-induced liver damage.

Systemic injection of products of activated neutrophils and H2O2 in myeloperoxidase-immunized rats leads to necrotizing vasculitis in the lungs and gut.

The strong association of anti-neutrophil cytoplasmic antibodies with various forms of systemic vasculitis suggests a role for these autoantibodies in the pathophysiology of systemic vasculitis. In the present study, we tested the hypothesis that release of neutrophil lysosomal enzymes in the presence of an anti-myeloperoxidase (anti-MPO) immune response may underlie the development of systemic vasculitis. Brown Norway rats were immunized with MPO in complete Freund's adjuvant or complete Freund's adjuvant alone. Two weeks after immunization, rats bad developed antibodies to human and rat MPO as measured by enzyme-linked immunosorbent assay. Next, rats were intravenously infused with 400 micrograms of a human neutrophil lysosomal extract containing 200 micrograms of MPO followed by 0.5 ml of a 1 mmol/L solution of H2O2 through a cannula inserted into the right jugular vein. Rats were sacrificed at 4 hours, 24 hours, 7 days, or 14 days, and several organs (lungs, heart, liver, spleen, gut, and kidneys) were examined for vasculitic lesions and inflammatory cell infiltrates. Macroscopically, patchy hemorrhagic spots were observed in the lungs and gut of MPO-immunized rats at days 7 and 14 after systemic infection of the neutrophil lysosomal extract and H2O2. Such changes were not observed at earlier time points or in control immunized rats. Histologically, the lungs of MPO-immunized rats sacrificed at days 7 and 14 showed patchy inflammatory cell infiltrates associated with vasculitis, granuloma formation, giant cells, and foci of hemorrhage. At 14 days, early signs of fibrosis were found with deposition of collagen and proliferation of fibroblasts. Furthermore, a prominent leukocytoclastic vasculitis was found in the small intestine of these rats characterized by fibrinoid necrosis and an extensive neutrophilic infiltrate. No inflammatory changes were found in the other organs studied (heart, liver, spleen, and kidneys). Control immunized rats, sacrificed at days 7 and 14 showed only some small foci of inflammatory infiltrates in the lungs whereas no inflammatory changes were found in the gastrointestinal tract. These studies show that release of products from activated neutrophils in the presence of anti-MPO autoantibodies may be relevant to the pathogenesis of anti-MPO-associated vasculitides.

Modulation of glomerular ECTO-ADPase expression by oestradiol. A histochemical study.

The effect of 17-beta-oestradiol (OE2) upon the activity of the glomerular anti-thrombotic ecto-enzyme ADPase was studied in cyclic and ovariectomized (OVX) Wistar rats. On day 0 (i.e. at the time of ovariectomy or 11 days after ovariectomy) rats received OE2-releasing Silastic implants or empty implants and were sacrificed on day 3, 10 or 21. Cryostat kidney sections were histochemically stained for ecto-ADPase activity using enzyme-histochemistry and glomerular reaction product was quantitatively evaluated by computerized image analysis. Both the histological distribution of reaction product in each glomerulus, as reflected by the relative glomerular area covered with reaction product, as well as enzyme activity, as reflected by staining intensity of the reaction product, were scored. The results show significantly decreased histological distribution after OVX; OVX, however, did not change enzyme activity. It further appeared that OE2 (partly) prevented the decrease of histological distribution in OVX rats, while the enzyme activity was significantly increased by exogenous OE2. In cyclic rats, OE2 did not change histological distribution, although OE2 significantly increased enzyme activity in these rats. It is concluded that glomerular ecto-ADPase expression in the rat kidney is influenced by one or more ovarian factor(s), a very likely candidate being oestradiol. These results may thus point to a dual action of OE2 upon haemostasis: In addition to the known enhancement of procoagulatory plasma factors by OE2, also anti-aggregatory effects may be stimulated by OE2 as reflected by upregulation of vessel wall associated ecto-ADPase activity.

A physiologic function for alkaline phosphatase: endotoxin detoxification.

Alkaline phosphatase (AP), a common enzyme present in many species including humans, has been studied extensively. Although the enzyme is routinely applied as a marker for liver function, its biologic relevance is poorly understood. The reason for this is obvious: the pH optimum of AP in vitro, as measured with the usual test substrates (+/-10.5), greatly exceeds the physiologic pH range as it occurs in biologic tissues. We now hypothesize that this relatively high pH optimum in vitro is related to dissociation of acidic groups in the protein preparation, which leads to the formation of negatively charged groups in the vicinity of the active site of the enzyme. These negatively charged groups may promote the activity of AP. We examined the possibility that endotoxin is a natural substrate for this enzyme because this phosphorylated substance is able to supply multiple negatively charged residues in the microenvironment of the enzyme at a physiologic pH level. Phosphate groups in the endotoxin molecule are known to be essential for the biologic activities of this bacterial product. The present study demonstrates that in intestinal and renal tissue specimens in vitro, AP is endowed with endotoxin dephosphorylating activity at pH levels closer to the physiologic range. This is also illustrated by our experiments in vivo showing that the toxicity of endotoxin is significantly reduced after exposure to AP preparations, as tested by inducing a local intradermal inflammatory reaction in rats. Collectively, our data suggest that the ubiquitous enzyme AP may accomplish protection against endotoxin, an equally ubiquitous product of Gram-negative bacteria that may cause lethal complications after an infection with these micro organisms.

Autoantibodies to myeloperoxidase aggravate mild anti-glomerular-basement-membrane-mediated glomerular injury in the rat.

Autoantibodies to myeloperoxidase (MPO) are present in sera from patients with various forms of vasculitis-associated glomerulonephritis. Evidence for a pathogenic role of anti-MPO antibodies has been provided mainly by in vitro studies. We studied the pathogenic role of autoantibodies to MPO in a rat model of mild immune-mediated glomerular injury. Brown Norway rats were immunized with human MPO in complete Freund's adjuvant or with complete Freund's adjuvant alone. At 2 weeks after immunization, rats had developed antibodies to human and rat MPO as detected by indirect immunofluorescence, enzyme-linked immunosorbent assay, and immunoprecipitation. At this time point, rats were intravenously injected with a subnephritogenic dose of 150 micrograms of rabbit anti-rat GBM. Rats were sacrificed at 4 hours, 24 hours, 4 days, and 10 days after antibody administration. Control immunized rats developed mild glomerulonephritis characterized by slight proteinuria at day 10 (14.8 +/- 8.1 mg/24 hours) and moderate intraglomerular accumulation of ED1+ macrophages. Crescent formation, tuft necrosis, and tubular atrophy were not observed in those rats. In contrast, rats immunized with MPO developed severe glomerulonephritis characterized by the early occurrence of severe hematuria, marked proteinuria at day 10 (76.2 +/- 18.2 mg/24 hours), and massive glomerular deposition of fibrin. Complement and rat IgG were present in insudative lesions, but no linear pattern along the glomerular capillary wall was observed. By light microscopy, severe glomerular lesions were found at day 10 consisting of crescent formation and fibrinoid necrosis of capillary loops. In the interstitium, tubular necrosis and atrophy and marked interstitial mononuclear infiltration were found in conclusion, autoantibodies to MPO severely aggravate subclinical anti-GBM disease demonstrating their in vivo pathogenic potential.

Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats.

Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GBM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these proteolytic enzymes along the glomerular capillary wall may induce glomerular injury. Here, we investigated the effects of PR3 and elastase on the induction of proteinuria and glomerular injury after renal perfusion of these enzymes in Brown-Norway rats. Perfusion of active elastase induced a dose-dependent proteinuria 24h after perfusion, while inactivated elastase did not. Perfusion of comparable amounts of active PR3 did not induce proteinuria. Light and electron microscopy showed no morphological abnormalities in any experimental group. However, immunohistology revealed that proteinuria occurring after perfusion of active elastase was associated with a strong reduction in intraglomerular expression of the heparan sulphate side chain and, to a lesser extent, of the protein core of heparan sulphate proteoglycans (HSPG). In vitro, both elastase and PR3 digested HSPG. However, PR3 bound to a lesser extent to HSPG than elastase. We conclude that elastase, but not PR3, induces proteinuria after in vivo renal perfusion. This differential effect probably relates to different binding to the GBM of those enzymes due to differences in their isoelectric points. Degradation of heparan sulfate proteoglycans, leading to the disappearance of their side chains that contribute to the polyanionic structure of the GBM, appears to be involved in the induction of proteinuria after perfusion of elastase.