Experimental autoimmune cystitis: further characterization and serum autoantibodies.

Previously, we described an animal model for interstitial cystitis (IC), experimental autoimmune cystitis (EAC) [Luber-Narod et al. Urol Res 24:367]. Further characterization of animals with EAC indicates that peak and mean urinary frequency are elevated compared with sham-injected controls and that the disease progresses with at least two cycles of exacerbations and remissions. We had shown evidence suggesting EAC to be autoimmune in nature. In this paper, we identify serum autoantibodies from 9/10 EAC animals which bind to a protein specific to rat bladder with a relative molecular weight of 12-kDa. Such autoantibodies are absent in 12/13 normal and sham-injected animals as well as animals which fail to develop EAC despite disease induction. These findings suggest that EAC is a reproducible model of cyclical increases of urinary frequency, and that a 12-kDa antigen is the target of autoantibodies which correlate with those elevations. Identification of this target antigen may explain the pathogenesis of increased urinary frequency in these animals and potentially in IC as well.

Agonist induced conformation alteration of neurotensin receptor and the mechanism behind Na+ inhibition of 125I-NT binding.

In the absence of Na+, 125I-Neurotensin (125I-NT) binding to the Neurotensin receptor (NTR) produces a stable noncovalent 125I-NT-NTR complex whose dissociation rate is extremely low even after the addition of 1 microM NT, 100 microM SR48692 (antagonist), 100 microM GPPNHP or 100 mM NaCl. Lowering the medium pH to 4.5 enhances the process (approximately 70% in 10 minutes). Labeling by photoactivatable 125I-Tyr3-Azo4-NT identifies a approximately 50 KD Mr band along with several other minor components. Interestingly, the labeling intensity is drastically reduced when binding is performed in the presence of Na+ or GPPNHP. However, a minor reduction is noticed when Na+ or GPPNHP is added to the medium after binding. The binding kinetics indicates that Na+ lowers the rate of 125I-NT association by acting as a noncompetitive inhibitor. On the contrary, Na+ favors the interaction of antagonist, SR48692 by lowering the value of Ki. GTPgamma35S binding to membranes in the presence of 30 mM NaCl suggests that Na+ inhibition of 125I-NT binding is due to the uncoupling of NTR associated G protein(s). In order to explain the entire phenomenon, a two-step, binding model has been proposed. In Step-1, interaction between NT and NTR produces a transient complex, which attains a stable state in the absence of NaCl via step-2, thereby altering the native NTR conformation. The presence of Na+ prevents step-2 by dissociating the transition complex.

Role of substance P in several models of bladder inflammation.

Substance P (SP) is a peptide found in the sensory nervous system which has multiple biologic effects including stimulation of muscle contraction, pain nociception, immune cell functions, plasma extravasation and a constellation of inflammatory effects. Here we investigate the role of SP in several animals models of bladder inflammation. Using the female Lewis rat, inflammation was induced using either xylene, lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (polyIC). Inflammation occurred rapidly (4 h) and was maintained in each model for at least 7 days. Each of these protocols decreased the bladder content of immunoreactive SP by approximately 50%, suggesting enhanced release. There was no change in the urinary frequency of these animals over 3 weeks, suggesting that urinary frequency changes are not mediated by acute inflammation. We also found that the SP receptor (NK1) antagonist, (-)CP96345, was unable to block the inflammation produced by polyIC, suggesting that SP is not an obligatory mediator of immune cell stimulation in this model.

17 beta-estradiol induced prostatitis in the rat is an autoimmune disease.

PURPOSE: Studies suggest that alteration in steroid hormone levels may be one of the factors causing nonbacterial prostatitis (NBP) in rats. We hypothesized that hormonally induced prostatitis in the rat may be an autoimmune disease. Studies were carried out to prove this hypothesis. MATERIALS AND METHODS: We injected 17 beta-estradiol (E2; 250 micrograms/kg. subcutaneously) or vehicle into 1-year-old male rats for 30 days, and isolated and cultured the splenocytes in the presence of con-A (Experiment 1). Approximately 10(7) splenocytes were adoptively transferred into young naive syngeneic rats. To find out whether or not the inflammation is mediated by T-lymphocytes, a pure population of T-lymphocytes from E2-treated 3-month-old rats was injected into young naive syngeneic rats (Experiment 2). To rule out the possibility that the inflammation was due to con-A itself, we cross-linked some T-cells with anti-CD3 antibody before adoptive transfer (Experiment 2). RESULTS: The recipients of splenocytes from E2-treated animals presented evidence of inflammation in terms of lymphocytic infiltration and presence of degranulated mast cells. Furthermore, we observed in these animals an increase in histamine-releasing peptide (HRP) levels, an indication of plasma extravasation. The T-cells stimulated by anti-CD3 antibody produced a similar degree of inflammation, thereby ruling out the possibility that the inflammation was due to con-A. The results also indicated that the immune response to antigen(s) is not dependent on the age of the animal but is dependent on a population of CD3+ T-cells. CONCLUSION: Our results demonstrate that hormonal imbalance and autoimmunity in male rats produce the symptoms of nonbacterial prostatitis.

Oxalate toxicity in LLC-PK1 cells, a line of renal epithelial cells.

PURPOSE: The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells. MATERIALS AND METHODS: Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed. RESULTS: Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations ( > 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells. CONCLUSIONS: Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Oxalate toxicity in LLC-PK1 cells: role of free radicals.

Oxalate, the most common constituent of kidney stones, is an end product of metabolism that is excreted by the kidney. During excretion, oxalate is transported by a variety of transport systems and accumulates in renal tubular cells. This process has been considered benign; however, recent studies on LLC-PK1 cells suggested that high concentrations of oxalate are toxic, inducing morphological alterations, increases in membrane permeability to vital dyes and loss of cells from the monolayer cultures. The present studies examined the basis for oxalate toxicity, focusing on the possibility that oxalate exposure might increase the production/availability of free radicals in LLC-PK1 cells. Free radical production was monitored in two ways, by monitoring the reduction of nitroblue tetrazolium to a blue reaction product and by following the conversion of dihydrorhodamine 123 (DHR) to its fluorescent derivative, rhodamine 123. Such studies demonstrated that oxalate induces a concentration-dependent increase in dye conversion by a process that is sensitive to free radical scavengers. Specifically, addition of catalase or superoxide dismutase blocked the oxalate-induced changes in dye fluorescence/absorbance. Addition of these free radical scavengers also prevented the oxalate-induced loss of membrane integrity in LLC-PK1 cells. Thus it seems likely that free radicals are responsible for oxalate toxicity. The levels of oxalate that induced toxicity in LLC-PK1 cells (350 microM) was only slightly higher than would be expected to occur in the renal cortex. These considerations suggest that hyperoxaluria may contribute to the progression of renal injury in several forms of renal disease.

Experimental autoimmune cystitis in the Lewis rat: a potential animal model for interstitial cystitis.

To develop an autoimmune animal model for interstitial cystitis (IC), we injected rats with Freund's adjuvant (CFA) containing bladder homogenate (experimentals) or CFA alone (shams). We observed a doubling of urinary frequency in the experimental animals over the shams (P = 0.004) and histopathologic changes (venular congestion) consistent with IC. Statistically significant bladder capacity changes were not found. Mast cell (MC) number was not statistically different between experimentals and controls but the number of MCs from section to adjacent section within the same animal's bladder did vary markedly, indicating the MC counts are not a reliable measure of disease in the rat bladder. Splenocytes cultured from the experimental animals and transferred to naive syngeneic recipients were capable of transferring the urinary frequency changes and vascular congestion while splenocytes from animals which did not develop the condition were without effect. In summary, we have developed and autoimmune model for IC consistent with the clinical features of IC. The features of this model can be transferred to naive syngeneic recipients via adoptive splenocyte transfer. The model will permit us to ask and answer important questions about the pathogenesis and treatment of the human disease.

Oxalate-induced damage to renal tubular cells.

Our own studies and those of others have shown that the incidence of calcium oxalate stones and plaques is markedly increased by nephrotoxins. The possible role of oxalate as a nephrotoxin has not been fully appreciated. However, recent studies in experimental animals and in cultured cells support this possibility. The results of these studies led us to hypothesize that hyperoxaluria promotes stone formation in several ways: by providing a substrate for the formation of the most common form of renal stones, calcium oxalate stones, and by inducing damage to renal epithelial cells. Damaged cells in turn would produce an environment favorable for crystal retention and provide membranous debris that promotes crystal nucleation, aggregation and adherence. The present report summarizes evidence for oxalate nephrotoxicity and discusses the potential importance of oxalate toxicity in the pathogenesis of stone disease.

Substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide.

The neuropeptide, substance P (SP), can stimulate secretion of TNF-alpha from macrophages. Neuroglia have SP receptors and subserve various macrophage-like functions in the central nervous system. We investigated whether SP stimulates secretion of TNF-alpha from primary cultures of neuroglial cells containing both astrocytes (approximately 90%) and microglia (approximately 10%). SP alone had no effect; however in the presence of LPS (10 ng/ml), SP (1 to 10 nM) caused a dose-dependent increase in TNF-alpha secretion above the level measured in response to LPS alone. The effective doses of SP correlated with 125I-labeled Bolton Hunter-conjugated SP binding (Kd 0.2 nM) to these cultures. Incubation with LPS did not change the number or affinity of SP-binding sites. In cultures enriched for microglia (> 99% pure), LPS stimulated the secretion of TNF-alpha but SP caused no enhancement. Microglia have no detectable 125I-labeled Bolton-Hunter-conjugated SP binding sites in the presence or absence of LPS. These results indicate that the action of SP is mediated through astrocytes. We investigated whether IL-1 mediates the SP enhancement of TNF-alpha secretion. Addition of IL-1-neutralizing antisera to mixed cultures stimulated with both LPS and 10 nM SP decreased TNF-alpha secretion to the level observed with LPS alone. LPS alone stimulated the secretion of IL-1 in a dose-dependent manner in the primary cultures, and this LPS-mediated IL-1 secretion was enhanced by SP. This enhancement was not observed in microglial cultures. SP may therefore play a role in neuropathologies in which these cytokines have been implicated.

An iodinated vasopressin (V1) antagonist blocks flank marking and selectively labels neural binding sites in golden hamsters.

An arginine-vasopressin (AVP) derivative, [d(CH2)5,Sar7]AVP (SAVP), has been characterized as an antagonist to vasopressin V1 receptors. Using AVP-dependent flank-marking behavior as a bioassay, it was possible to verify that iodinated SAVP (I-SAVP) retains biological activity within the central nervous system, as the antagonist blocked the behavior. Furthermore, 125I-SAVP was used to localize specific V1 binding sites in the brain. The resulting binding was localized to discrete anatomical sites, and highly specific to V1-like receptors. While we confirmed previous findings using 3H-AVP in golden hamsters, we also identified binding in many areas previously unreported (e.g., arcuate and paraventricular nuclei of the hypothalamus, tenia tecta, posteromedial cortical nucleus of the amygdala, and zona incerta), suggesting that 125I-SAVP provides a greater level of resolution. In addition, specific binding was observed in the lateral septum, anterior hypothalamus, and midbrain central gray, areas that have previously been shown to trigger flank marking in response to AVP microinjection. The presence of AVP binding sites in limbic and mesencephalic areas involved in the regulation of flank marking suggests that this neuropeptide may play an important role as a neurotransmitter at multiple levels in the neural circuits controlling this behavior.

Guanine nucleotides decrease the affinity of substance P binding to its receptor.

The inhibitory effect of guanine nucleotides on the binding of 125I-Bolton-Hunter conjugated substance P (125I-BHSP) to rat submaxillary gland membranes has been confirmed and further explored. The evidence presented here indicates that this is due to a marked loss of binding affinity. In the presence of 5'-guanylyl imidodiphosphate (GppNHp) there is (1) a greater than or equal to 20-fold increase in the Kd as determined by Scatchard analysis and (2) the concentration of SP required to inhibit half of the 125I-BHSP binding (IC50) increased approximately 30-fold. Consistent with a marked decrease in affinity is an approximately 100-fold increase in the rate of dissociation of 125I-BHSP following addition of GppNHp. Complete restoration of high-affinity binding was achieved by removal of the guanine nucleotide.

Isolation and identification of a polypeptide in the Hsp 70 family that binds substance P.

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.

Immune system associated antigens expressed by cells of the human central nervous system.

HLA-DR, HLA-DQ and HLA-DP are class II major histocompatibility complex (MHC) antigens necessary for T cell binding and antigen presentation. Interleukin-2 is a lymphokine used by the immune system to signal proliferation of cells in the immune response. Using unfixed tissue and free-floating immunohistochemistry, we show profuse immunoreactivity for these immune antigens in white and gray matter samples from normal and Alzheimer's (AD) disease patients. On morphologic grounds, immunoreactive cell types appear to include astrocytes, microglia, macrophages, and endothelial cells or pericytes.

Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease.

HLA-DR is a class II major histocompatibility complex antigen which in the periphery confers antigen presenting capability. We have previously shown that this marker is profusely expressed in cortex of elderly and Alzheimer's disease (AD) patients, as is the receptor for the lymphokine interleukin-2. We now report presence of additional immune-related antigens in AD, and distributional differences from normal elderly controls. In gray matter, HLA-DR immunoreactivity is normally sparse, except in AD where it co-localizes with virtually all neuritic plaques. HLA-DR positive T cells can be demonstrated in Alzheimer's disease brain tissue, as can instances of apposition between putative brain microglia and T cells. In addition, cells with the morphologic characteristics of astrocytes label for natural killer cell antigen (Leu-11), and apparent lymphocytes bearing T helper and T cytotoxic/suppressor cell antigens are observed. These and other data suggest that the glial proliferation and scavenger activity characteristic of Alzheimer's disease may occur in an immune context and may play an important role in the pathogenesis of the disorder.