Pedro L. Vera

Inhibition of Macrophage Migration Inhibitory Factor or Its Receptor (CD74) Attenuates Growth and Invasion of DU-145 Prostate Cancer Cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.

Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells

BackgroundThe importance of various inflammatory cytokines in maintaining tumor cell growth and viability is well established. Increased expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) has previously been associated with various types of adenocarcinoma.MethodsMIF IHC was used to localize MIF in human bladder tissue. ELISA and Western blot analysis determined the synthesis and secretion of MIF by human bladder transitional cell carcinoma cells. The effects of MIF inhibitors (high molecular weight hyaluronate (HA), anti-MIF antibody or MIF anti-sense) on cell growth and cytokine expression were analyzed.ResultsHuman bladder cancer cells (HT-1376) secrete detectable amounts of MIF protein. Treatment with HA, anti-MIF antibody and MIF anti-sense reduced HT-1376 cell proliferation, MIF protein secretion, MIF gene expression and secreted inflammatory cytokines. Our evidence suggests MIF interacts with the invariant chain, CD74 and the major cell surface receptor for HA, CD44.ConclusionsThis study is the first to report MIF expression in the human bladder and these findings support a role for MIF in tumor cell proliferation. Since MIF participates in the inflammatory response and bladder cancer is associated with chronic inflammatory conditions, these new findings suggest that neutralizing bladder tumor MIF may serve as a novel therapeutic treatment for bladder carcinoma.

Afferent and sympathetic innervation of the dome and the base of the urinary bladder of the female rat

The afferent and sympathetic innervation of different regions of the urinary bladder (bladder dome vs. bladder base) was examined in the female rat using simultaneous injections of two fluorescent tracers. Retrogradely labeled cells were found in the dorsal root ganglia (DRG; L1-L3 and L6-S1), the sympathetic chain (SC; T12-L6), the inferior mesenteric ganglia (IMG) and the major pelvic ganglia (MPG). There were very few double-labeled cells, indicating that the dome and the base of the bladder receive innervation (afferent or sympathetic) from separate and distinct neuronal populations. Most of the sympathetic innervation of the bladder arose from the SC (dome: 77%; base: 89%) and it was carried equally by the hypogastric and pelvic nerves. The distributions of SC postganglionic neurons innervating the dome and the base of the bladder were very similar. In contrast, the contribution of IMG neurons was almost entirely restricted to the dome of the bladder (22%), with less than 1% innervating the base. Tyrosine hydroxylase-immunoreactive (TH) neurons in the MPG displayed a strong sexual dimorphism. Many TH neurons were found in the male MPG, but very few in the female MPG. In the female, these TH neurons projected almost exclusively to the bladder base of the female rat and were responsible for 10% of the sympathetic innervation of the base. Less than 1% innervated the dome. Therefore, prevertebral ganglia (IMG and MPG) show a strong regional selectivity in the innervation of the bladder of the female rat. The possible functional implications of this organization are discussed.

Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer

BackgroundMacrophage migration inhibitory factor (MIF) is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum) levels.Recent studies from another laboratory failed to document these results. This studys aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIFs receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate.MethodsMIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis.ResultsMedian serum MIF amounts were significantly elevated in prostate cancer patients (5.87 ± 3.91 ng/ml; ± interquartile range; n = 115) compared with patients with no documented diagnosis of prostate cancer (2.19 ± 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected in culture medium from prostate cancer cell lines (LNCaP and PC-3).ConclusionIncreased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer.

Cyclophosphamide-Induced Cystitis Increases Bladder CXCR4 Expression and CXCR4-Macrophage Migration Inhibitory Factor Association

Background Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis. Methods and Findings Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3rd day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations. Conclusions These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.

Macrophage Migration Inhibitory Factor Is Upregulated in an Endotoxin-Induced Model of Bladder Inflammation in Rats

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine found in epithelial cells as preformed stores, such that MIF release can activate innate immune responses. Our identification of MIF stores in the urothelium suggests that MIF may function in the bladders initial response to infectious stimuli, such as lipopolysaccharide (LPS). To test this hypothesis, we observed changes in MIF, cyclooxygenase-2 (COX-2) and c-fos in the bladder, L6-S1 spinal cord, dorsal root ganglion (DRG), and major pelvic ganglion (MPG) and MIF changes in the prostate following intravesical LPS. Intravesical LPS induced bladder edema and leukocyte infiltration, as well as increased MIF protein and mRNA in the bladder and lumbosacral spinal cord. Expression of immediate-early gene c-fos, a transcription factor used as a marker of neuronal activation, increased in the L6-S1 spinal cord and L6-S1 DRG of rats that received LPS. We conclude that significant increases in bladder MIF expression and protein in response to intravesical LPS may represent part of this organs initial innate immune response. In addition, MIF upregulation may represent a neural response to visceral inflammation. Finally, changes in prostate MIF content after intravesical LPS suggest that MIF may be involved in viscerovisceral interactions associated with chronic pelvic pain syndromes.

Macrophage Migration Inhibitory Factor is Increased in the Urine of Patients With Urinary Tract Infection: Macrophage Migration Inhibitory Factor-Protein Complexes in Human Urine

PURPOSE MIF is a proinflammatory cytokine present in preformed stores in human urothelium. In animal models of bladder inflammation, including bacterial cystitis, MIF is up-regulated in the bladder and released from the bladder as a high molecular weight complex. We compared urine MIF amounts in patients with UTI to that in patients without UTI, and we examined and identified MIF-protein complexes in urine. MATERIALS AND METHODS Using enzyme-linked immunosorbent assay we compared MIF levels in the urine of 14 patients with UTI to levels in 16 controls with no UTI. Western blotting under native, denaturing and reducing conditions was done to examine MIF complexes found in urine. Mass spectrometry identified MIF associated proteins in urine, while co-immunoprecipitation confirmed the associations. RESULTS Mean urine MIF amounts +/- SEM determined by enzyme-linked immunosorbent assay were significantly greater in 14 patients with UTI compared to that in 16 controls (1.96 +/- 0.40 vs 0.59 +/- 0.09 ng/mg creatinine, p <0.01). Western blotting under denaturing conditions showed several high molecular weight complexes (100 to 165 kDa) that increased in UTI urine as well as typical, monomeric MIF (12 kDa). Mass spectrometry identified associated MIF proteins, including ceruloplasmin, albumin and uromodulin. Co-immunoprecipitation confirmed mass spectrometry findings and also identified MIF interaction with alpha-2-macroglobulin. CONCLUSIONS Increased urine MIF amounts in patients with bacterial cystitis support our experimental evidence showing a role for MIF in pelvic visceral inflammation. The novel finding of an association of MIF with other urine proteins suggest that the physiologically relevant form of MIF may be an MIF-protein complex.

Separate urinary bladder and external urethral sphincter neurons in the central nervous system of the rat: Simultaneous labeling with two immunohistochemically distinguishable pseudorabies viruses.

This work examines the distribution, in the central nervous system, of virus-labeled neurons from the rat urinary bladder and the external urethral sphincter simultaneously within the same tissue sections. Two immunohistochemically distinct pseudorabies virus strains were injected into male Sprague--Dawley rats (approximately 280 g). One virus was injected into the bladder and the other into the external urethral sphincter. After incubation intervals of 2, 2.5 and 3 days, sections from the spinal cord and brain were treated immunohistochemically to detect cells which were labeled separately by each virus or were labeled by both viruses. The major result of these experiments is that each strain of virus labeled a separate population of neurons and that some neurons were labeled by both strains. In the lumbosacral cord, 3 days post-infection, neurons labeled by virus from the external urethral sphincter were found in Onufs nucleus, the dorsal gray commissure, and the superficial dorsal horn. Neurons labeled by virus from the urinary bladder were found in the L6--S1 and L1--L2 spinal cord segments within the dorsal gray commissure, the intermediolateral area and the superficial dorsal horn. Double-labeled interneurons were mainly located in the dorsal gray commissure although some were also found in the intermediolateral area and the superficial dorsal horn. In the medulla, external urethral sphincter neurons and bladder neurons and double-labeled neurons were found in the reticular region and the raphe. More rostrally, bladder neurons were located in the pontine micturition center and external urethral sphincter neurons were found in the locus coeruleus and subcoeruleus. A very small number of double-labeled neurons were found in the pontine micturition center and the locus coeruleus or subcoeruleus.

Anatomical evidence for two spinal 'afferent-interneuron-efferent' reflex pathways involved in micturition in the rat: a 'pelvic nerve' reflex pathway and a 'sacrolumbar intersegmental' reflex pathway.

We labeled interneurons in the L1-L2 and L6-S1 spinal cord segments of the rat that are involved in bladder innervation using transneuronal retrograde transport of pseudorabies virus (PRV) in normal animals and in animals with selected nerve transections. Preganglionic neurons were identified using antisera against choline acetyltransferase (ChAT). In some experiments we labelled parasympathetic preganglionic neurons (PPNs) in the L6-S1 spinal cord by retrograde transport of Fluorogold from the major pelvic ganglion. We identified bladder afferent terminals using the transganglionic transport of the anterograde tracer cholera toxin subunit b. We present anatomical evidence for two spinal pathways involved in innervation of the bladder. First, in the intact rat, afferent information from the bladder connects, via interneurons in L6-S1, to the PPNs that provide the efferent innervation of the bladder. The afferent terminals were located mainly in close apposition to interneurons located dorsal to the retrogradely labeled PPNs. Second, using L6-S1 ganglionectomies or L6-S1 ventral root rhizotomies we limited viral transport to the sympathetic pathways innervating the bladder. This procedure also labelled interneurons (but not PPNs) with PRV in the L6-S1 spinal cord in a location very similar to those described in the intact rat. These interneurons also receive bladder afferent terminals but we propose that they project to sympathetic preganglionic neurons, most of which are in the L1-L2 spinal segments. Based on this anatomical evidence, we propose the existence of two spinal reflex pathways involved in micturition: a pathway limited to a reflex arc in the pelvic nerve (presumably excitatory to the detrusor muscle); and a pathway involving the pelvic nerve and sympathetic nerve fibers, some of which may travel in the hypogastric (presumably inhibitory to the detrusor muscle).

Anatomical location of Macrophage Migration Inhibitory Factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat

BackgroundPrevious work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat.ResultsIn the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons.ConclusionsTherefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIFs expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes.