Lactobacillus rhamnosus strain JB-1 reverses restraint stress-induced gut dysmotility.

BACKGROUND: Environmental stress affects the gut with dysmotility being a common consequence. Although a variety of microbes or molecules may prevent the dysmotility, none reverse the dysmotility. METHODS: We have used a 1 hour restraint stress mouse model to test for treatment effects of the neuroactive microbe, L. rhamnosus JB-1™ . Motility of fluid-filled ex vivo gut segments in a perfusion organ bath was recorded by video and migrating motor complexes measured using spatiotemporal maps of diameter changes. KEY RESULTS: Stress reduced jejunal and increased colonic propagating contractile cluster velocities and frequencies, while increasing contraction amplitudes for both. Luminal application of 10E8 cfu/mL JB-1 restored motor complex variables to unstressed levels within minutes of application. L. salivarius or Na.acetate had no treatment effects, while Na.butyrate partially reversed stress effects on colonic frequency and amplitude. Na.propionate reversed the stress effects for jejunum and colon except on jejunal amplitude. CONCLUSIONS & INFERENCES: Our findings demonstrate, for the first time, a potential for certain beneficial microbes as treatment of stress-induced intestinal dysmotility and that the mechanism for restoration of function occurs within the intestine via a rapid drug-like action on the enteric nervous system.

Spatiotemporal maps reveal regional differences in the effects on gut motility for Lactobacillus reuteri and rhamnosus strains.

BACKGROUND: Commensal bacteria such as probiotics that are neuroactive acutely affect the amplitudes of intestinal migrating motor complexes (MMCs). What is lacking for an improved understanding of these motility effects are region specific measurements of velocity and frequency. We have combined intraluminal pressure recordings with spatiotemporal diameter maps to analyze more completely effects of different strains of beneficial bacteria on motility. METHODS: Intraluminal peak pressure (PPr) was measured and video recordings made of mouse ex vivo jejunum and colon segments before and after intraluminal applications of Lactobacillus rhamnosus (JB-1) or Lactobacillus reuteri (DSM 17938). Migrating motor complex frequency and velocity were calculated. KEY RESULTS: JB-1 decreased jejunal frequencies by 56% and 34% in colon. Jejunal velocities increased 171%, but decreased 31% in colon. Jejunal PPr decreased by 55% and in colon by 21%. DSM 17938 increased jejunal frequencies 63% and in colon 75%; jejunal velocity decreased 57%, but increased in colon 146%; jejunal PPr was reduced 26% and 12% in colon. TRAM-34 decreased frequency by 71% and increased velocity 200% for jejunum, but increased frequency 46% and velocity 50% for colon; PPr was decreased 59% for jejunum and 39% for colon. CONCLUSIONS & INFERENCES: The results show that probiotics and other beneficial bacteria have strain and region-specific actions on gut motility that can be successfully discriminated using spatiotemporal mapping of diameter changes. Effects are not necessarily the same in colon and jejunum. Further research is needed on the detailed effects of the strains on enteric neuron currents for each gut region.

Nerve growth factor expression and release in allergic inflammatory disease of the upper airways.

It is well known that allergic airways disease is characterized by inflammation and hyperresponsiveness, but the link between these two conditions has not been elucidated. We have previously shown that in allergic rhinitis, hyperresponsiveness is attributable to increased neural reactivity. We thus hypothesized that nerve growth factor (NGF), which is expressed by inflammatory cells and effects changes that lead to increased neural responsiveness, could be a pivotal mediator in this disease. Using reverse transcription-polymerase chain reaction (RT-PCR), Western immunoblotting, and ELISA to evaluate NGF expression and release, we found that subjects with allergic rhinitis have significantly decreased NGF mRNA in superficial nasal scrapings and significantly higher baseline concentrations of NGF protein in nasal lavage fluids, compared with control subjects. Nasal provocation with allergen significantly increased NGF protein in nasal lavage fluids of subjects with allergic rhinitis, but not of control subjects. The concentrations of NGF protein in nasal lavage fluids were not affected by provocation with the vehicle for allergen or with histamine. These data provide the first evidence of a steady state of dysregulation in mucosal NGF expression and release in allergic rhinitis, and support a role of this neurotrophin in the pathophysiology of allergic inflammatory disease of the human airways.

Nerve growth factor and neuroimmune interactions in inflammatory diseases.

Discovered almost 50 years ago, nerve growth factor (NGF) has been extensively studied in various biological systems. NGF has recently been suggested to play an important role in mediating and/or regulating immune response, in addition to its trophic and tropic effects on nerve growth and regeneration It is clear that in complex interactions between immune cells and nervous system NGF plays a central role. We have only just begun to identify and understand the direct mechanisms by which NGF activates target cells, the precise identity of the target cells, and the particular factors released from target cells. Nerve growth factor together with possibly other neurotrophins such as BDNF (brain-derived nerve growth factor), GDNF (glial-derived nerve growth factor) or NT3 are important modulators of immunity. More detailed studies are needed at the receptor, mediator and cellular levels to better understand the neuroimmunomodulatory properties of neurothrophins and NGF. The nature of the involvement of NGF in inflammation and inflammatory diseases remains a particularly interesting question. By blocking NGF or mediators released upon NGF activation, we are able to control the progress of inflammation, thereby opening many therapeutic opportunities for the future.

Neural hyperresponsiveness and nerve growth factor in allergic rhinitis.

BACKGROUND: In allergic rhinitis, symptoms are triggered not only by allergens but also by environmental irritants. Hereinafter we address the hypothesis that this is reflective of increased responsiveness of the neural apparatus which, in turn, may be attributable to upregulation of nerve growth factor (NGF) in this disease. METHODS: We compared subjects with active allergic rhinitis and healthy volunteers in terms of sensitivity and/or magnitude of three nerve-mediated responses, namely (1) the sneezing reflex induced by histamine, (2) the central or nasonasal reflex depicted by contralateral secretions induced by unilateral nasal challenge with capsaicin, and (3) the axonal reflex depicted by plasma extravasation upon capsaicin challenge. We have also measured NGF levels in nasal lavage fluids at baseline and with allergen provocation in rhinitis and healthy subjects. RESULTS: Compared to healthy individuals, subjects with active allergic rhinitis were found to have (1) significantly greater sensitivity and reactivity of the sneezing reflex, (2) significantly greater secretory responsiveness to sensory nerve stimulation, and (3) significantly greater plasma extravasation indicated by albumin leakage following capsaicin nasal challenge. We also found that subjects with active allergic rhinitis have significantly greater baseline levels of NGF in nasal lavage fluids compared to their healthy counterparts, and that these levels can be increased by allergen nasal provocation. CONCLUSION: The responsiveness of the neural apparatus of the nose is significantly greater in patients with active allergic rhinitis. The increased presence of NGF in the nasal mucosa of these patients supports the hypothesis that this neurotrophin may be implicated in neural hyperresponsiveness.

Nerve growth factor and cytokines mediate lymphoid tissue-induced neurite outgrowth from mouse superior cervical ganglia in vitro.

Superior cervical ganglia (SCG) from neonatal mice were cultured with adult murine lymphoid tissue explants in Matrigel (Collaborative Biomedical, Bedford, MA). After 1 and 2 days in culture, many neurites grew toward thymus and spleen. Normal mesenteric lymph node (MLN) induced a smaller effect; however, activated MLN (isolated from mice 10 days after infection with Nippostrongylus brasiliensis; Nb-MLN-10d) caused significantly increased neurite outgrowth. To determine the roles of nerve growth factor (NGF) and cytokines in the promotion of neuritogenesis by lymphoid tissues, anti-NGF and various anti-cytokines were added to cocultures. Anti-NGF inhibited most of the neurite outgrowth toward thymus and spleen but only partially that toward Nb-MLN-10d. Anti-mouse IL-1 beta also significantly reduced the number of neurites growing toward thymus, spleen, and normal MLN. The number of neurites growing toward Nb-MLN-10d was significantly reduced by anti-IL-1 beta, anti-IL-3, anti-IL-6, or anti-GM-CSF. Exogenous IL-1 beta and IL-3 caused neurite outgrowth in single SCG cultures; and the IL-1 beta-, but not the IL-3-, mediated effect was completely blocked by anti-NGF. In one-day thymus/SCG cocultures, endogenous IL-1 was not detectable at concentrations sufficient to cause nerve growth; however, ample NGF was present in the thymic tissues and culture supernatants, but not in SCG. These data suggest that IL-1 mediates NGF production in lymphoid tissues, which in turn induces the growth of sympathetic nerves. Moreover, IL-3, IL-6, or GM-CSF produced during inflammation might also play important roles in the stimulation of nerve growth in vivo.

Stress-induced murine abortion associated with substance P-dependent alteration in cytokines in maternal uterine decidua.

Stress is known to induce abortions, but underlying mechanisms are unknown. Both alloimmunization and injection of antibody to the asialoGM1 determinant of natural killer cells have been shown to prevent stress-triggered abortion in mice. DBA/2J-mated CBA/J female mice were used to investigate the influence of stress during early gestation on systemic hormone levels and on cytokines in the decidua that are thought to be relevant to abortion in nonstress-related murine abortion. Lowered levels of progesterone did not occur as a result of stress. In stressed mice, increased levels of the abortogenic cytokine tumor necrosis factor alpha (TNF alpha) were associated with decreased levels of pregnancy-protective transforming growth factor beta 2-related suppressive activity in uterine decidua. In the alloimmunized animals where stress failed to boost the abortion rate, these effects were abrogated. Production of TNF alpha may be stimulated by the neurotransmitter substance P (SP); after injection of an SP receptor antagonist or SP-antibody, stress failed to increase the abortion rate above the background level. The increased levels of TNF alpha we observed in the stressed animals were completely abrogated in the animals that had received the SP receptor antagonist; stress also failed to decrease the pregnancy-protective suppressive activity in the decidua of these animals. The data indicate that stress may inhibit protective suppressor mechanisms and promote secretion of abortogenic cytokines such as TNF alpha via neurotransmitter SP.

Substance P induces ion secretion in mouse small intestine through effects on enteric nerves and mast cells.

We used genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice and congenic WBB6F1 +/+ normal (+/+) mice to examine the role of mast cells in substance P-induced intestinal ion secretion. Isolated sheets prepared from segments of the midportion of the small intestine were studied in Ussing chambers. Substance P caused a dose-dependent increase in short-circuit current (Isc) that was approximately 50% less in intestine from W/Wv than from +/+ mice. Similar results were obtained for substance P-(4-11) (the COOH terminus) and substance P methyl ester [a selective neurokinin (NK)-1 agonist]. Histamine H1 or H2 antagonists reduced the Isc responses to substance P in intestine from +/+ mice but had no effect in intestine from W/Wv mice. In addition, reconstitution of intestinal mast cells in W/Wv mice by intravenous injection of +/+ bone marrow cells normalized the tissues' secretory responses to substance P or substance P methyl ester. However, in W/Wv and +/+ mice, the selective NK1 antagonist CP-96345 virtually abolished intestinal responses to substance P, and the responses were also markedly inhibited by neural blockade with tetrodotoxin. In contrast, in tetrodotoxin-pretreated intestine, histamine antagonism caused a further reduction in the responses to substance P only in +/+ mouse tissues. Taken together, our results suggest that the effects of substance P on intestinal Isc KN1 receptors but that the neuropeptide acts via effects on enteric nerves and mast cells. The data thus support the concept that mast cells and enteric nerves participate in the regulation of substance P-induced intestinal ion secretion.

Neuroimmunomodulation: classical and non-classical cellular activation.

As neuroimmunologists, we are often faced with the fact that some substances can either enhance or inhibit particular immune/inflammatory cell functions. This 'duality' could only partially be explained by dose-dependency and the fact that in a variety of systems, heterogenous cell populations are commonly used. For example it has been repetitively shown that cell proliferation, immunoglobulin synthesis and NK (natural killer) activity could be enhanced, inhibited or not affected at all by such neuropeptides as somatostatin (SOM) or vasoactive intestinal peptide (VIP), depending on the experimental conditions. Even substance P (SP), which, in general, stimulates lymphocyte activity, can, under certain conditions, possess an inhibitory activity. These apparent discrepancies between various groups and experimental conditions met with a strong reservation among 'classical' immunologists as they questioned the true physiological role that neuro-immune interactions play in normal and disease states. However, upon a detailed analysis of the data, it become obvious why such discrepancies abounded. Not only are we comparing totally different responses in different species, but almost always we compare different experimental conditions. In lieu of this, the reproducibility of the experiments within the same laboratory is in fact very high. One fundamental and striking observation is the fact that at the level of a homogeneous cell population, a differential response could be evoked by the same neuropeptide over a range of concentrations. For the purpose of this brief report we will focus on the cellular responses to the neuropeptide substance P and we will try to illustrate why such differential responses are possible. Some of the physiological data relating to the effects of SP on cell function will be discussed. This will be followed by a synopsis of SP receptor mechanisms on effector cells and finally the mechanism by which SP activates secondary messenger systems in these cells.

Substance-P-mediated intestinal inflammation: inhibitory effects of CP 96,345 and SMS 201-995.

The proinflammatory peptide substance P (SP) has been shown to be intimately involved in the local inflammatory processes of Trichinella-spiralis-induced murine intestinal inflammation. Significant increases in SP, increased myeloperoxidase levels coupled with local morphological deterioration of the jejunum and impaired lymphocyte responses to exogenous SP in vitro have been associated with the model. We have recently determined that the elimination of increased levels of SP via anti-SP antibody therapy can spare the murine gastrointestinal tract much of the pathologies associated with the parasitic infection. Here we further demonstrate that the somatostatin analogue SMS 201-995 as well as the SP receptor antagonist CP 96,345 can effectively decrease the inflammation and lost lymphocyte function seen in the jejunum of T. spiralis-infected mice. Again, both intestinal morphology and myeloperoxidase levels were shown to return to normal values upon treatment. The above results suggest that SP is an important modulator of gastrointestinal inflammation.

Neuronal factors modulating immunity.

In this minireview we will discuss some evidence suggesting that the immune response is under neuronal regulation. In particular, we will concentrate on the effects that various neuropeptides have on immunity both in vitro and in vivo. Of these, vasoactive intestinal peptide, substance P, somatostatin, and calcitonin gene related peptide will be discussed in detail. In addition, the effects of nerve growth factor on the immune system will be presented. Finally, a possible role for these neuropeptides in various diseases and its clinical relevance will be suggested.

Hormones and local immunity.

The modulatory role of hormones in the regulation of the immune response has been well documented. Here, we present some thoughts on how sex hormones affect immunity, particularly at mucosal sites. We further discuss the possible pitfalls and difficulties associated with analysis of the data.

Interleukin 1 beta-induced increase in substance P in rat myenteric plexus.

BACKGROUND: Substance P (SP) is increased in the inflamed intestine of Trichinella spiralis-infected rats, but the underlying mechanism is unknown. Interleukin 1 beta (IL-1 beta) messenger RNA and protein is expressed in the longitudinal muscle-myenteric plexus (LM-MP) of this model. Thus, the purpose of the study was to examine the ability of human recombinant IL-1 beta (hrIL-1 beta) to increase SP in LM-MP preparations from the intestine of noninfected rats. METHODS: LM-MP preparations were incubated with hrIL-1 beta, and immunoreactive SP (IR-SP) was assessed in the tissues by radioimmunoassay or immunohistochemistry. RESULTS: hrIL-1 beta increased IR-SP in the tissue in a time- and concentration-dependent manner, being maximal after 6 hours at a concentration of 10 ng/mL. The IR-SP could be depleted by scorpion venom, and immunohistochemistry revealed increased staining for SP within nerves of the LM-MP. The action of IL-1 beta was dependent on protein synthesis, was receptor mediated, and was not due to endotoxin contamination of the cytokine preparation. CONCLUSIONS: hrIL-1 beta stimulates the synthesis of SP in myenteric nerves of rat intestine.

Intestinal permeability in allergic rats: nerve involvement in antigen-induced changes.

In vivo uptake of the probe 51Cr-labeled EDTA from the jejunum of egg albumin (EA)-sensitized rats was compared with controls at baseline and after intraluminal antigen challenge. Probe recovery in blood was 60-80% greater in sensitized animals during the baseline period, suggesting that sensitization resulted in increased intestinal permeability. Sensitized, but not control, rats demonstrated a 15-fold increase in 51Cr-EDTA uptake after intraluminal antigen; no change occurred with an unrelated protein. Macromolecular recovery was also enhanced in sensitized animals, since serum levels of immunoreactive EA were elevated 14-fold compared with controls. Antigen challenge was accompanied by biochemical (protease release) and morphological (reduced numbers) evidence of mast cell degranulation in sensitized rats. The neurotoxin tetrodotoxin (applied directly to ligated jejunal segments) inhibited EA-induced uptake of 51Cr-EDTA and antigen. In isolated jejunum from sensitized rats, tetrodotoxin reduced secretory responses to luminal, but not serosal, antigen. These results indicate that neural factors may influence the uptake of molecules from the gut lumen during intestinal anaphylaxis.

Inhibition of murine intestinal inflammation by anti-substance P antibody.

Several neuropeptides have recently been shown to affect various aspects of the inflammatory process. Among these, the neuropeptide substance P possesses a host of immune modifying actions, which include the enhancement of lymphocyte activity, macrophage function, and neutrophil chemotaxis. The role of substance P during inflamed states has, as yet, not been fully described. Here, in T. spiralis-infected mice, we parallel increased levels of substance P both locally, (the gut) and peripherally (serum) with decreased lymphocyte responsiveness. Upon the introduction of in vivo antisubstance P antibody during the infection, levels of substance P, gastrointestinal inflammation, and lymphocyte proliferation are significantly restored to baseline (noninfected) levels. These findings suggest that the neuropeptide substance P plays an important role in promoting inflammation. It also offers the basis for future pharmacological interventions.

Expression of nerve growth factor receptor immunoreactivity on follicular dendritic cells from human mucosa associated lymphoid tissues.

Nerve growth factor (NGF) was originally considered as a trophic factor for peripheral sympathetic and sensory neurones; however, recent reports indicate that NGF may induce proliferation of immune and haematopoietic cells. Histochemical studies conducted in human spleen and lymph nodes have suggested the presence of NGF receptor (NGF-R) immunoreactive elements in secondary follicles; however the nature of the cells bearing the NGF-R in lymphoid tissue has not been determined. In this paper we report the results of an immunohistochemical study conducted on mucosa associated lymphoid tissue. Using a specific monoclonal antibody to human NGF-R (mAb 20.4) we observed an NGF-R-immunoreactive population in all secondary lymphoid follicles examined. Double immunostaining revealed that this population was composed of follicular dendritic cells (FDC); lymphoid cells within the germinal centres did not appear to be 20.4 immunoreactive. Cell suspensions from tonsillar follicles also contained NGF-R immunopositive dendritic cells which were enriched by a 20.4 labelled magnetic bead procedure, revealing cells with the morphological characteristics of FDC. Mononuclear cells from human peripheral blood did not contain any NGF-R-immunoreactive elements using our techniques.

Synoviocyte derived granulocyte macrophage colony stimulating factor mediates the survival of human lymphocytes.

Synoviocytes have been shown to be effector cells capable of synthesizing and secreting a variety of cytokines and growth factors. We demonstrate here that synoviocyte derived conditioned medium has immunoregulatory properties as it enhances human peripheral blood lymphocyte survival in a dose dependent manner in vitro. The effect elicited by synoviocyte derived conditioned medium from patients with rheumatoid arthritis (RA) was greater than that induced by synoviocyte derived conditioned medium from patients with osteoarthritis. Granulocyte-macrophage colony stimulating factor (GM-CSF) was found in synoviocyte derived conditioned medium with significantly higher levels present in synoviocyte derived conditioned medium from patients with RA. Recombinant human GM-CSF induced survival of human lymphocytes in vitro and a monoclonal antibody to human GM-CSF fully abrogated synoviocyte derived conditioned medium induced survival. Our results demonstrate that synoviocyte derived GM-CSF may be important in the retention of lymphocytes, which is a central pathological characteristic of the rheumatoid joint.

Are lymphocytes a target for substance P modulation in arthritis?

The contribution of the neuropeptide substance P to the pathogenesis of rheumatoid arthritis (RA) has recently been suggested. The presence of immunoreactive substance P in the serum and joint fluid of RA patients was significantly increased compared with age-matched control patients. To investigate the ability of substance P to alter lymphocyte activity during the disease, lymphocytes were isolated from the synovial fluid and blood of RA patients and their ability to respond to substance P as measured by [3H]thymidine uptake was characterized. Upon exposure of RA synovial fluid and peripheral blood lymphocytes to various concentrations of substance P in vitro, no increase in proliferation was witnessed. To the contrary, control peripheral blood lymphocyte proliferation was significantly enhanced by various concentrations of substance P. However, synoviocytes from the joints of RA patients were responsive to substance P stimulation. These data suggest that substance P receptors may be desensitized on systemic and local lymphocytes in RA, or the proinflammatory activities of substance P may be mediated via the synovial membrane during chronic inflammation.