Neuroimmune mechanisms in health and disease: 2. Disease.

In the second part of their article on the emerging field of neuroimmunology, the authors present an overview of the role of neuroimmune mechanisms in defence against infectious diseases and in immune disorders. During acute febrile illness, immune-derived cytokines initiate an acute phase response, which is characterized by fever, inactivity, fatigue, anorexia and catabolism. Profound neuroendocrine and metabolic changes take place: acute phase proteins are produced in the liver, bone marrow function and the metabolic activity of leukocytes are greatly increased, and specific immune reactivity is suppressed. Defects in regulatory processes, which are fundamental to immune disorders and inflammatory diseases, may lie in the immune system, the neuro endocrine system or both. Defects in the hypothalamus-pituitary-adrenal axis have been observed in autoimmune and rheumatic diseases, chronic inflammatory disease, chronic fatigue syndrome and fibromyalgia. Prolactin levels are often elevated in patients with systemic lupus erythematosus and other autoimmune diseases, whereas the bioactivity of prolactin is decreased in patients with rheumatoid arthritis. Levels of sex hormones and thyroid hormone are decreased during severe inflammatory disease. Defective neural regulation of inflammation likely plays a pathogenic role in allergy and asthma, in the symmetrical form of rheumatoid arthritis and in gastrointestinal inflammatory disease. A better understanding of neuroimmunoregulation holds the promise of new approaches to the treatment of immune and inflammatory diseases with the use of hormones, neurotransmitters, neuropeptides and drugs that modulate these newly recognized immune regulators.

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.

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.

Inhibitory effect of diazepam on human natural killer activity in vitro.

The in vitro effect of diazepam on natural killer (NK) cell activity isolated from human peripheral blood has been investigated. NK cell function was estimated by means of a radioactive chromium (51Cr) assay in which human erythroleukaemia K562 cells were used as the target. Diazepam suppressed NK cell function in a concentration-dependent manner (10(-7)-10(-5) M) and a positive correlation was found between the dose of diazepam and inhibition detected by percentage killing of K562 cells. These data indicate that diazepam influences cytotoxic activity and diminishes anti-viral and anti-tumour defence reactions in humans.

Immunomodulatory properties of diazepam-binding inhibitor: effect on human interleukin-6 secretion, lymphocyte proliferation and natural killer cell activity in vitro.

We have examined the influence of diazepam binding inhibitor (octadecaneuro-peptide, DBI33-50) on cell mediated immune responses including LPS-stimulated monocyte IL-6 secretion, PHA induced lymphocyte proliferation and NK cell function in humans. All studies were performed in vitro on isolated human peripheral blood mononuclear cells in the absence or presence of synthetic DBI33-50. It has been shown that DBI33-50, in concentration between 10(-6)-10(-8) M, enhances the LPS-induced secretion of IL-6, as determined by specific bioassay for this monokine. On the other hand DBI33-50 (10(-6)-10(-12) M), had no significant effect on either PHA-induced lymphocyte proliferation or NK cell function. This data suggests a possible immunomodulatory role for DBI33-50 as an endogenous neuropeptide, which stimulates IL-6 secretion by human monocytes.

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.

The effect of nerve growth factor on DNA synthesis, cyclic AMP and cyclic GMP accumulation by mouse spleen lymphocytes.

Nerve growth factor (NGF), a trophic neuropeptide, is known to stimulate development, and to be important in the maintenance and survival of sympathetic and sensory neurons. Considering the presence of specific receptors on the surface of spleen cells, the effect of 2.5s nerve growth factor on 3H-thymidine uptake, cAMP and cGMP accumulation in mouse spleen lymphocytes has been studied. It was found that NGF added in vitro at the concentrations between 4 x 10(-7) and 4 x 10(-8) M significantly inhibited the incorporation of 3H-thymidine into lymphocytes DNA and increased cAMP levels in a dose-dependent manner but had no effect on cGMP levels. The maximal stimulation of cAMP synthesis occurred between 5 and 30 min after the NGF addition to the culture medium. When NGF was administered in vivo a significant dose-dependent inhibition of the lymphocytes proliferation was observed. These results indicate that an early increase of cAMP concentration is responsible for the antiproliferative action of NGF on mouse spleen lymphocytes and suggest that NGF could play an important role in the regulation of immune system function.

Selective modulation of the natural killer activity of murine intestinal intraepithelial leucocytes by the neuropeptide substance P.

Neuropeptides can influence immune effector cell function at both systemic and mucosal immune sites. We examined the ability of substance P (SP) to modulate the natural killer (NK) activity of intestinal intraepithelial leucocytes (IEL). Yac-1 killing by IEL but not splenic cells was increased after either 18 hr preincubation or 6 hr of co-incubation with SP. We also examined the NK activity of IEL and spleen isolated from mice treated with SP in vivo. The selective increase in NK activity of IEL occurred without any demonstrable change in the number or phenotype of the IEL. The IEL responsive to SP in vivo and induced in vivo by SP were both Thy-1- and did not kill the NK insensitive mastocytoma cell line P815. Lastly, we examined the ability of SP to induce the release of interleukin-2 (IL-2) and IL-4 from IEL after 6 and 18 hr of in vitro culture. No increase in the release of these cytokines was observed, suggesting that IL-2 and IL4 are not involved in the local augmentation of IEL NK activity by SP. These observations suggest that SP has a selective stimulatory effect on intestinal activity and may play a role in the regulation of intestinal cell-mediated immunity.

Human lung fibroblast-derived granulocyte-macrophage colony stimulating factor (GM-CSF) mediates eosinophil survival in vitro.

Tissue eosinophilia has been reported to occur in pulmonary fibrosis, a disease characterized by chronic inflammation and lung fibroblast proliferation. We have examined the in vitro interaction of these two cell types by determining the in vitro survival of human peripheral blood eosinophils co-cultured with human lung fibroblasts. Survival of eosinophils cultured alone was 10% at day 3 and less than 1% at day 7. In contrast, survival of eosinophils that had been co-cultured with fibroblasts was 98, 90, 73, and 69% at days 3, 7, 10, and 14, respectively. Fibroblast-conditioned medium (CM) elicited a similar result in a dose-dependent fashion. Survival of eosinophils cultured with CM which had been preincubated with a monoclonal-neutralizing antibody to human GM-CSF was inhibited in a dose-dependent manner. Human recombinant-derived GM-CSF supported eosinophil survival in the dose-dependent fashion. Survival at day 7 of eosinophils treated with one single dose of GM-CSF (10 U/ml) was 64%. The effect of fibroblast-CM on eosinophils likely represents true survival since eosinophil proliferation as determined by [3H]thymidine incorporation did not occur. We also report that freshly isolated eosinophils had normal ultrastructural, scanning and transmission electron microscopy characteristics, and were normodense. In contrast, eosinophils co-cultured for 7 days with fibroblasts acquired irregular shapes and became hypodense and partially degranulated. Thus, our results indicate that human lung fibroblast-derived GM-CSF mediates the in vitro survival of human eosinophils.(ABSTRACT TRUNCATED AT 250 WORDS)

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit.

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors.

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.

Neuropeptides and immunity.

Our studies have clearly shown that neuropeptides have a profound effect on immunoglobulin synthesis both in vivo and in vitro. The effects varied according to the neuropeptide added or the tissue from which the lymphocytes were obtained. Substance P caused the most pronounced enhancement of both functions, especially in Peyer's patch cells, where it selectively increased IgA synthesis. Somatostatin was inhibitory, and the effect of vasoactive intestinal peptide varied according to the source of the cells. We have previously shown that neuropeptides also cause mast cell secretion and that only substance P was effective in this regard on intestinal mucosal mast cells. Therefore, we looked for microanatomic relationships between peptidergic nerves and immune effector cells. Mast cells appear to have structural associations with neuropeptides-containing nerves in the intestine. Nerve growth factor, known to promote the growth of sensory afferent and sympathetic nerves, has significant direct effects on mast cells. In vitro, this substance caused enhanced antigen mediated histamine release and, in vivo, extensive mast cell hyperplasia. Also, in humans, we were able to produce increased numbers of mast cell/basophil colonies from peripheral blood in the presence of nerve growth factor.

The differential effect with time of neuropeptides on the proliferative responses of murine Peyer's patch and splenic lymphocytes.

The neuropeptides substance P (SP), somatostatin (SOM), and vasoactive intestinal peptide (VIP) have been shown to modulate lymphocyte DNA, RNA, and immunoglobulin synthesis. We have previously shown that SP enhances while SOM and VIP inhibit proliferation of murine splenic and Peyer's patch lymphocytes when cells were cultured with concanavalin A and neuropeptides for 72 h. Here we show that the effect of neuropeptides, in particular SP, is dependent on the amount of time that lymphocytes are incubated with NP. We found that SOM and VIP always inhibited cell proliferation for incubation times of 2 to 72 h. In contrast when cells were exposed to SP for 24 h or less, there was inhibition of [3H]thymidine uptake by both Peyer's patches and splenic lymphocytes. Significant enhancement in DNA synthesis by lymphocytes from both organs was only seen when cells were incubated with SP for the whole 72 h. We believe that our data may explain some of the conflicting reports regarding the effects of neuropeptides on cell proliferation.