How should bladder sensation be measured? ICI-RS 2011.

AIMS: Disturbed bladder sensations, or in broader terms, sensory dysfunctions are increasingly recognized as key elements in the origin and manifestation of symptom syndromes of urinary dysfunction. Adequate assessment of bladder sensation is crucial to improve our understanding of the pathophysiology and treatment of urinary dysfunction. This manuscript summarizes the discussions of a think tank on "How to measure bladder sensation" held at the ICI-RS meeting in 2011. METHODS: Based upon literature reviews on bladder sensation presented at the think tank in the ICI-RS meeting, discussions evolved which were summarized in the ICI-RS report. Different physicians/researchers further elaborated on this report, which is presented in this manuscript. RESULTS: Bladder sensations are not merely the result of bladder distension. Other factors inside the bladder or bladder wall: central processing and/or cognitive manipulation may play an important role. Current methods to measure sensations such as urodynamics, voiding diaries, forced diuresis, electrical stimulation and brain imaging are likely sub-optimal as they only consider part of these factors in isolation. CONCLUSIONS: Different methods to measure bladder sensations have been described and are used in clinical practice. Current methods only address part of the parameters responsible for the generation and perception of urinary sensations. Further focused research is required, and several recommendations are provided.

Lessons learned: impact of a continence promotion activity for older community-dwelling women.

AIMS: Few studies have documented the effectiveness of continence promotion programs targeting older incontinent women. We sought to evaluate the impact of an interactive continence workshop on changing participants' attitudes, knowledge and skills in relation to self-managing or seeking care for incontinence. METHODS: A quasi-experimental prospective cohort study with repeated measures was carried out on a population of 90 incontinent women aged 55-87 participating in a continence promotion workshop. Inclusion criteria were a weekly average of one or more episodes of involuntary urine loss during the preceding 3 months and having never sought help for this problem. Incontinence-related knowledge, attitudes, skills and intentions for seeking care were assessed immediately prior and subsequent to the workshop. Three- and 6-month telephone follow-ups were conducted to determine rates of healthcare seeking and reasons for not seeking care. RESULTS: Improvements in incontinence-related knowledge and attitudes occurred in up to 94% participants. Forty-three percent of the study participants initiated and were satisfied with self-treatment, and an additional 42% consulted a health care professional. CONCLUSION: Interactive continence workshops promote self-management and consultation seeking among older women with incontinence. Further testing of different strategies for promoting continence awareness needs to occur in larger studies with more sensitive instruments, a control group, and better specification of the goals, process and outcomes of the health promotion activity being tested.

Trospium chloride has no effect on memory testing and is assay undetectable in the central nervous system of older patients with overactive bladder.

BACKGROUND: Muscarinic receptors in the brain play an important role in cognitive function, especially memory, and there is growing awareness that specific antimuscarinic drugs for overactive bladder (OAB) may have adverse central nervous system (CNS) effects. Selection of an antimuscarinic OAB drug with reduced potential for CNS effects could be especially beneficial in the elderly people, in whom even the modest cognitive impairment may negatively affect independence. PURPOSE: The purpose of the study is to determine if trospium chloride is assay detectable in the CNS of older adults with OAB and to assess whether deterioration of memory occurs in these individuals. METHODS: Twelve cognitively intact older adults (>or=65-75 years old) with OAB were given extended-release trospium chloride 60 mg once daily over a 10-day period to achieve plasma steady-state levels. Standardised memory testing (Hopkins Verbal Learning Test-Revised and Brief Visuospatial Memory Test-Revised) was performed predose and postdose. Cerebrospinal spinal fluid (CSF) and plasma samples were drawn on day 10 and assayed for trospium chloride. Predose (day 0) and postdose (day 10) results on the memory tests were compared using a reliable change index to assess a meaningful change in learning or memory. RESULTS: Trospium chloride levels in all the CSF samples (n = 72) of all participants were assay undetectable (<40 pg/ml) on day 10 at steady-state peak plasma concentration concurrent with measureable peak plasma values (C(max) = 925 pg/ml). Repeat memory testing revealed no significant net drug effect on learning or recall. CONCLUSIONS: This is the first study to investigate for the presence of an OAB antimuscarinic in the human brain, performed by assaying for concentrations of trospium chloride and correlating with simultaneous clinical cognitive safety measures. The results of both pharmacological and neuropsychological testing support the hypothesis of a lack of detectable CNS penetration for the quaternary amine trospium chloride.

A simple apparatus for performing short-time (1--2 seconds) uptake measurements in small volumes; its application to D-glucose transport studies in brush border vesicles from rabbit jejunum and ileum.

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10--20 microliter. Using this technique the kinetic parameters Km and V of D-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the Km for D-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.

The T cell death knell: immune-mediated tumor death in renal cell carcinoma.

The antitumor effect of T cells is executed either through CD95 or Perforin (PFN)/Granzyme B (GrB) pathways. Induction of apoptosis by either mode requires activation of caspase family members. However, recent studies have suggested that cell death can proceed in the absence of caspase induction and apoptotic events. We investigated the contribution of CD95 and PFN/GrB-mediated cytotoxicity to apoptotic and necrotic mechanisms of cell death in human renal cell carcinoma. Although freshly isolated and cultured tumors expressed CD95 on their surface, they were resistant to CD95-mediated apoptosis. CD95 resistance coincided with decreased levels of FADD protein and diminished caspase-3-like activity. In contrast, we demonstrated that tumor cell death mediated by PFN/GrB can be achieved in the absence of functional caspase activity and is accompanied by a dramatic accumulation of nonapoptotic necrotic cells.

Tumor-induced sensitivity to apoptosis in T cells from patients with renal cell carcinoma: role of nuclear factor-kappaB suppression.

Antitumor immunity fails to adequately develop in many cancer patients, including those with renal cell carcinoma (RCC). A number of different mechanisms have been proposed to explain the immune dysfunction observed in cancer patient T cells. Here we show that T cells from RCC patients display increased sensitivity to apoptosis. Tumor-infiltrating lymphocytes (TILs) display the most profound sensitivity, because 10-15% of those cells are apoptotic when assessed by terminal deoxynucleotidyltransferase-mediated nick end labeling in situ, and the number of apoptotic TILs further increases after 24 h of culture. Peripheral blood T cells from RCC patients are not directly apoptotic, although T lymphocytes derived from 40% of those individuals undergo activation-induced cell death (AICD) upon in vitro stimulation with phorbol myristate acetate and ionomycin. This is in contrast to T cells from normal individuals, which are resistant to AICD. TILs and peripheral blood T cells from RCC patients also exhibit impaired activation of the transcription factor, nuclear factor (NF)-kappaB. Additional findings presented here indicate that the heightened sensitivity of patient T cells to apoptosis may be tumor induced, because supernatants from RCC explants sensitize, and in some instances directly induce, normal T cells to apoptosis. These same supernatants also inhibit NF-kappaB activation. RCC-derived gangliosides may represent one soluble tumor product capable of sensitizing T cells to apoptosis. Pretreatment with neuraminidase, but not proteinase K, abrogated the suppressive effects of tumor supernatants on both NF-kappaB activation and apoptosis. Additionally, gangliosides isolated from tumor supernatants not only inhibited NF-kappaB activation but also sensitized T cells to AICD. These findings demonstrate that tumor-derived soluble products, including gangliosides, may contribute to the immune dysfunction of T cells by altering their sensitivity to apoptosis.

Interferon-gamma and CXC chemokine induction by interleukin 12 in renal cell carcinoma.

Interleukin 12 (IL-12) is known to play an important role in the development of an antitumor response. Its activity has been shown to be dependent upon the intermediate production of IFN-gamma and the influx into the tumor of CD8 lymphocytes. In a murine model, tumor regression induced by IL-12 treatment correlated with IFN-gamma, IP-10, and Mig expression in the tumor bed and was abrogated by antibodies to both chemokines. Here we examined the effects of rHuIL-12 on IFN-gamma and CXC chemokine gene expression in patients with renal cell carcinoma (RCC) in an attempt to determine whether a similar series of molecular events leading to IL-12-mediated tumor regression in mice is also detectable in humans. As in the murine RENCA model, cultured RCC cells themselves could be induced by IFN-gamma to synthesize IP-10 and Mig mRNA. Explanted RCC produced IFN-gamma and IP-10 mRNA in response to IL-12 treatment, which was consistent with the finding that biopsied RCC tumors from IL-12-treated patients also variably expressed augmented levels of those molecules after therapy. Although Mig mRNA was present in the majority of biopsied tumors prior to treatment, both the Mig and IP-10 chemokines as well as IFN-gamma were induced in the peripheral blood mononuclear cells of IL-12-treated patients. Skin biopsies of IL-12-treated patients also all synthesized IP-10 mRNA. This study demonstrates that recombinant human IL-12 therapy of patients with RCC has the potential to induce the expression of gene products within the tumor bed that may contribute to the development of a successful antitumor response.

Mechanisms of apoptosis in T cells from patients with renal cell carcinoma.

Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results from several laboratories suggest that FasL (L/CD95L) expression in tumors may be responsible for this process. In this study of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional because in coculture experiments, FasL+ tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an effective T-cell-mediated antitumor response.

A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes.

We previously reported the isolation and characterization of cDNA clones encoding novel lipopolysaccharide (LPS)-inducible mRNAs from murine peritoneal macrophages. We now present the complete coding sequence of a cDNA previously termed D3. Analysis of multiple clones from a murine macrophage cDNA library provided a complete cDNA sequence of approximately 1.6 kb. The corresponding RNA contains a single open reading frame encoding a hydrophilic protein composed of 425 amino acids and is characterized by a region including three perfect and two imperfect repeats of a seven-amino-acid sequence. Based on nucleotide and deduced amino acid sequence, this mRNA is a new member of a previously described multigene cluster of interferon-inducible genes termed the Mouse 200 series genes. This new sequence most closely resembles gene 204 because both D3 and 204 genes have segments containing the seven-amino-acid repeat sequence. The Mouse 202 and 204 genes, however, have an approximately 200-amino-acid carboxyl-terminal domain that is absent in the LPS-inducible macrophage-derived cDNA. In addition, D3, 202, and 204 can all be distinguished from one another by virtue of unique 3' noncoding regions 200-300 base pairs in length. The D3 unique sequence is largely restricted to the smallest of the three size classes of this gene family expressed in macrophages and is not detected in interferon- or platelet-derived growth factor-stimulated fibroblasts. Overall, three separate mRNAs have now been described, each of which has three or more of a possible seven nucleotide sequence domains. Although the function(s) of the members of this gene family remains unknown, the multiple forms inducible by diverse stimuli and their restricted cell type expression suggest diverse and important physiologic roles for their products in inflammation.

Differential protein synthesis by murine peritoneal macrophages elicited by various stimuli.

Protein synthetic patterns of murine peritoneal macrophages were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of 35S methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory, and activated macrophages had numerous common features that distinguished them from the other normal non-macrophage cell types examined, unique proteins also characterized each macrophage population from the others. The accumulation by resident macrophages of proteins 23, 25, and 37 distinguished them from elicited cells, as did the former's more abundant synthesis of proteins 54 and 52. The protein synthetic patterns of inflammatory thioglycollate- and proteose peptone-elicited macrophages were strikingly similar, save for the former's greater levels of accumulation of proteins 14 and 28, and the latter's more pronounced expression of p23.5. Peritoneal macrophages elicited by treatment with heat-killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes, and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16-h or 72-h functional assays. They shared a common protein synthetic profile that differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages. These tumoricidal macrophages were unique in synthesizing a protein(s) of approximate molecular weight 26,000 daltons. A time-course study employing P. acnes-activated peritoneal macrophages indicated that p26 accumulation decayed with tumoricidal capacity as a function of time in culture, although no direct correlation between lytic activity and p26 expression could be definitively established. Peritoneal macrophages elicited with proteose peptone were not directly tumoricidal but were rendered so upon in vitro treatment with nanogram amounts of bacterial lipopolysaccharide. The accumulation of low levels of p26 by the newly explanted proteose peptone-elicited macrophages suggests the possibility that this protein characterizes macrophage populations primed as well as triggered for tumoricidal activity.

Tissue-specific expression of murine IP-10 mRNA following systemic treatment with interferon gamma.

We have examined the tissue distribution of 10-kd inflammatory protein (IP-10) mRNA expression in C57Bl/6 mice injected intravenously (i.v.) with various inflammatory stimuli. IP-10 mRNA was strongly induced by interferon-gamma (IFN-gamma) in liver and kidney but only poorly in skin, heart, and lung. IFN-gamma had nearly equivalent access to these tissues as indicated by the distribution of radiolabeled recombinant IFN-gamma 1 h after injection. The time course of IP-10 mRNA appearance was rapid and transient in both liver and kidney; maximal expression in the liver (2 h) preceded that in the kidney (3 h) and declined rapidly thereafter in both tissues. Expression of IP-10 mRNA in the liver and kidney was highly sensitive to IFN-gamma treatment; nearly maximal stimulation occurred with injection of 500 U of IFN-gamma per mouse. Comparable stimulation of IP-10 mRNA expression in splenic macrophages required 10,000 U of IFN-gamma administered i.v., indicating that liver and kidney responses are 10- to 20-fold more sensitive. IP-10 mRNA expression in both tissues was not restricted to stimulation by IFN-gamma but was also seen with injection of lipopolysaccharide (LPS) (25 micrograms/mouse) or IFN-beta (100,000 U/mouse). Two other members of the IP-10 gene family, KC (gro) and JE (MCP-1), were expressed at lower levels under similar treatment conditions. Analysis of IP-10 mRNA distribution in the liver and kidney by in situ hybridization indicated that expression in both tissues was most prominent in the reticuloendothelial cell system, particularly in the endothelial lining of the microvascular circulation. Although the function of the IP-10 gene product has not been defined, these results suggest that it may play an important role in the response of both the liver and kidney to systemic inflammation.

Immune-inflammatory mechanisms in IFNgamma-mediated anti-tumor activity.

IFNgamma is a functionally pleiotropic cytokine which shows considerable potency in promoting anti-tumor functions in vivo. Despite limited efficacy when delivered systemically either to experimental animals or patients, IFNgamma appears to play an important and perhaps critical role in directing the development of immune-mediated tumor destruction when expressed within the tumor bed. This has been demonstrated both by use of tumor cells transduced to express IFNgamma and by the use of IL-12 which is able, at least is murine models, to promote an IFNgamma-dependent, T cell mediated anti-tumor response. Recent studies indicate that the therapeutic efficacy of IFNgamma in tumor models depends critically upon the ability of the tumor cells themselves to respond to IFNgamma. Though IFNgamma is able to induce anti-viral activity and has direct anti-proliferative effects on some tumor cell lines, immunomodulatory function also appears to be an important component of its anti-tumor action. This is mediated through the action of several different classes of IFNgamma-inducible gene expression which control antigen processing and presentation, leukocyte trafficking, and indirect tumor cytotoxicity.

LPS regulation of specific protein synthesis in murine peritoneal macrophages.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) analysis of biosynthetically labeled proteins of murine peritoneal macrophages elicited by inflammatory and activating stimuli indicated that the accumulation of a small number of cell-associated proteins was altered after in vitro treatment with bacterial lipopolysaccharide (LPS). Both increases and decreases in the accumulation of specific proteins were observed after LPS stimulation. Proteins of approximately 87, 43, 37, 30, and 28 Kd were similarly regulated by LPS in proteose peptone-, P. acnes-, and M. bovis BCG-elicited macrophages. Thioglycollate-elicited and resident peritoneal macrophages showed very few changes in the pattern of proteins synthesized after LPS treatment. Many of the proteins whose accumulation was increased by LPS in the elicited macrophages (proteins of approximately 87, 52, 43, 37, and 28 Kd) were already synthesized at high levels in resident macrophages. LPS stimulation also altered the accumulation of many of the same proteins in bone marrow-derived macrophages, indicating the lack of T lymphocyte influence on the LPS-induced changes in macrophages. LPS stimulation of highly purified B cells caused changes in the accumulation of several proteins of 70 and 78 Kd, which were different from those regulated by LPS in peritoneal macrophages.

Lipopolysaccharide-induced gene expression in murine peritoneal macrophages is selectively suppressed by agents that elevate intracellular cAMP.

Elevation of intracellular cAMP has been associated with the suppression of macrophage activation. The present study has examined the effects of agents that alter intracellular levels of cAMP on LPS-induced macrophage gene expression. Treatment of murine peritoneal macrophages with trace amounts of LPS leads to dramatically enhanced expression of multiple mRNA including the competence genes JE and KC, first observed in platelet-derived growth factor-stimulated fibroblasts, and those encoding the inflammatory monokines IL-1 and TNF. If macrophages are first treated with cholera toxin or dibutyryl cAMP 15 min before stimulation with LPS, the accumulation of mRNA encoding both JE and TNF is strongly suppressed whereas mRNA levels for KC and IL-1 are unaffected. The suppression of JE and TNF mRNA levels is dose dependent, in the range of 10 to 500 microM dibutyryl cAMP; concentrations as high as 1 mM do not affect the expression of either KC or IL-1. When dibutyryl cAMP is added to macrophages after initiation of LPS treatment, suppressive effects are diminished in a time-dependent fashion. Furthermore, dibutyryl cAMP suppresses the LPS-induced transcriptional activation of the TNF gene. Previous work has shown that the LPS-induced expression of JE appears to be mediated by hydrolysis of polyphosphoinositides and involves a post-transcriptional mechanism. Treatment with dibutyryl cAMP suppresses JE expression induced by treatment with phorbol ester and A23187 suggesting that inhibition of gene expression must act at a site other than the initial transmembrane signaling event. Finally, dibutyryl cAMP only marginally affects the constitutive transcription of the JE gene indicating that suppression may involve a post-transcriptional mechanism. These results indicate that expression of genes encoding inducible early proteins and inflammatory monokines are selectively regulated by elevation of intracellular cAMP. Such effects may be pleiotropic in nature involving multiple molecular mechanisms.

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages.

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.

IFN-gamma and lipopolysaccharide differentially modulate expression of tumor necrosis factor receptor mRNA in murine peritoneal macrophages.

Expression of TNF receptor (TNFR) mRNA has been examined in murine peritoneal macrophages stimulated with LPS and/or IFN-gamma. LPS markedly enhanced expression of a heterogenous population of mRNA, which hybridized with a cDNA encoding the type II TNFR. mRNA expression was optimally induced by 4 to 8 h and returned to baseline by 24 h after stimulation. Interestingly, though IFN-gamma can synergize with LPS for the expression of TNF-alpha, it abrogated the LPS-mediated enhancement of type II TNFR in a dose-dependent fashion. IFN-alpha, though less effective, had a qualitatively comparable effect. These effects were selective for the type II TNFR because levels of mRNA encoding the type I TNFR did not vary appreciably with any of the treatments described. The effects of IFN-gamma on LPS-mediated TNFR expression were dependent on the sequence of exposure; pretreatment with IFN-gamma was most effective at blocking response to LPS, whereas IFN-gamma added 1 h after initiation of LPS treatment had little or no effect. The effects of both LPS and IFN-gamma on type II TNFR expression were mediated at least in part by modulation of transcription. The effects of both LPS and IFN-gamma were also independent of protein synthesis because inclusion of cycloheximide in the treatment protocol did not abrogate either the inductive or the suppressive effects. These findings suggest that IFN-gamma and LPS modulate the physiologic action of TNF through complex mechanisms involving effects on the transcription of TNF-alpha itself and on receptors through which it may act in autocrine or paracrine fashion.

Antisecretory and antiinflammatory properties of bismuth subsalicylate.

The antisecretory properties of Pepto-Bismol (PB) and its active ingredient, bismuth subsalicylate (BSS), were studied in ligated (rabbit and pig) and unligated (rat) intestinal-segment models. When PB was administered to segments before intestinal inoculation with heat-labile (LT) Escherichia coli or Vibrio cholerae enterotoxins, the inhibition of fluid accumulation was 74%-94% and 60%-91%, respectively. In the pig, the percentages of inhibition by PB of fluid accumulation produced by organisms or toxins were 69% for E. coli P57 producing heat-stable enterotoxin (ST), 89%-95% for E. coli P155 producing ST and LT, 52% for ST alone, 95% for LT alone, and 73% for ST and LT. When PB was administered to the pig immediately after inoculation with E. coli P57, E. coli P155, ST alone, LT alone, or ST and LT, the percentages of inhibition of fluid accumulation were 76%, 80%, 56%, 97%, and 69%, respectively. However, in the rabbit and rat, PB failed to inhibit fluid accumulation when it was administered 5-60 minutes after inoculation of cholera or E. coli LT enterotoxins. In the rabbit the combination of BSS and the vehicle of PB was synergistic in preventing the fluid accumulation normally produced by cholera toxin. Finally, when PB or BSS was administered 30 minutes before intestinal inoculation with arachidonic acid in a rat model of inflammatory diarrhea, the percentages of inhibition of fluid accumulation ranged, in a dose-responsive fashion, from 16% to 113% for PB and from 25% to 111% for BSS.

Synergistic cooperation between T cell lymphokines for induction of the nitric oxide synthase gene in murine peritoneal macrophages.

The ability of T cell-derived cytokines to induce the expression of the nitric oxide synthase (NOS) gene in murine peritoneal macrophages was examined. IL-2 or TNF-alpha alone had no effect either on gene expression or enzyme activity, whereas IFN-gamma had only modest activity. When IL-2 or TNF-alpha were used in combination with IFN-gamma, there was a marked cooperative induction of both mRNA and enzyme activity. The cooperative effects were truly synergistic, as the consequences of combined cytokine treatment were many times greater than was seen with any of the agents acting independently. The expression of NOS mRNA and enzyme activity in response to combined lymphokine treatments was a continuous process reaching optimal levels between 24 and 48 h after stimulation. Concentration dependency for both IL-2 and TNF-alpha suggested that their effects were mediated through interaction with the corresponding defined cell surface receptors. Human rTNF-alpha was as effective a stimulus as murine TNF-alpha; because human TNF-alpha is recognized only by the p55 Type II TNF receptor, this structure appears to mediate the response to TNF-alpha. When IL-2 and TNF-alpha were added at saturating doses in the presence of IFN-gamma, there was an additive effect on NOS mRNA expression suggesting that IL-2 and TNF-alpha cooperate with IFN-gamma through at least partially distinct intracellular signaling pathways. Expression of NOS mRNA in response to IFN-gamma/IL-2 or IFN-gamma/TNF-alpha treatment required protein synthesis, suggesting that cooperative cytokine induction of NOS involves the intermediate expression of new gene products. Such molecular controls for regulation of inducible macrophage gene expression can be contrasted with regulatory control of other inflammatory genes such as IP-10 and TNF-alpha.

IFN-gamma and IFN-beta independently stimulate the expression of lipopolysaccharide-inducible genes in murine peritoneal macrophages.

We have recently described the isolation and characterization of a set of cDNA encoding genes whose expression is induced or enhanced in murine peritoneal macrophages by treatment with LPS. In the present report we have analyzed the expression of the mRNA which hybridize with these cDNA probes in macrophages treated with other cytokines known to modulate functional activity. Three distinct patterns of expression have been documented. Two genes (D3 and C7) are inducible by LPS, IFN-gamma, and IFN-beta; D3 is comparably sensitive to all three, whereas C7 is more sensitive to LPS and IFN-gamma than to IFN-beta. The mRNA encoded by D8 is expressed in response to LPS and IFN-beta but not in response to IFN-gamma. Finally the gene encoded by D5 is inducible only in cells treated with LPS. The expression of all three cytokine-inducible mRNA was both dose and time dependent and was mediated by increased transcriptional activity of the genes. As with stimulation by LPS, the expression induced by IFN was independent of protein synthesis and occurred in a rapid and transient fashion. TNF-alpha had little or no detectable effect on any of the genes by themselves. The expression of C7, however, could be induced synergistically by treatment with a combination of TNF-alpha and either IFN-gamma of IFN-beta. The expression of these genes was not specific for macrophages as both IFN were able to induce a comparable pattern of gene expression in BALB/c 3T3 cells. Treatment of macrophages with dexamethasone inhibited LPS-induced C7 and D8 expression but did not affect that seen in response to IFN-gamma or IFN-beta, respectively. The results suggest that IFN and LPS act to modulate early gene expression by the generation of at least three overlapping but distinct signaling pathways. In some cases the pathway(s) which mediate response to LPS appear to be mechanistically distinct from those which mediate response to IFN-beta or IFN-gamma. The spectrum of stimuli and cell types which express these and other early genes suggest that they may play an important role in orchestration of the inflammatory response.