Neurohormonal-cytokine interactions: implications for inflammation, common human diseases and well-being

The neuroendocrine system affects the immune system through the neuroendocrine humoral outflow via the pituitary, and through direct neuronal influences via the sympathetic, parasympathetic (cholinergic) and peptidergic/sensory innervation of peripheral tissues. Circulating hormones or locally released neurotransmitters and neuropeptides regulate major immune functions, such as antigen presentation, antibody production, lymphocyte activity, proliferation and traffic, and the secretion of cytokines including the selection of T helper (Th)1 or Th2 cytokine responses. During inflammation, the activation of the stress system, through induction of a Th2 shift protects the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones, substance P, ATP and the activation of the corticotropin-releasing hormone/substance P-histamine axis may actually facilitate inflammation, through induction of interleukin (IL)-1, IL-6, IL-8, IL-18, tumor necrosis factor (TNF)-alpha and CRP production. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic anti-inflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may play a role in the pathogenesis of atopic/allergic and autoimmune diseases, obesity, depression and atherosclerosis. Better understanding of the neuroendocrine control of inflammation may provide critical insights into mechanisms underlying a variety of common human immune-related diseases.     

Th1-Th2 response in hyperprolactinemic mice infected with Salmonella enterica serovar Typhimurium

Prolactin (PRL) is a pituitary hormone and a cytokine known to regulate several physiological functions. It plays a role in modulating the immune system of rodents and humans. A hormonal protection against listeria and salmonella infections has been previously ascribed to effects of PRL on immunocompetent cells. Here, the role of PRL in the Th1-Th2 response was evaluated based on the pattern of cytokines release by splenocytes from hyperprolactinemic mice infected with Salmonella enterica serovar Typhimurium. Hyperprolactinemia by pituitary graft reduced the number of bacteria in spleens of in vivo infected mice. Modulation of Th1 (IFN-gamma, IL-12) and Th2 (IL-4, IL-10) cytokine production by splenic cells was found. Our results indicate that PRL can up-regulate IFN-c and IL-12 secretion in response to salmonella infection, confirming its in vivo immunostimulatory effect and suggesting hormonal participation in the genesis and sustenance of the Th1 response.     

Immunoregulation of autocrine prolactin: Suppressing the expression of costimulatory molecules and cytokines in T lymphocytes by prolactin receptor knockdown

Ample evidence indicates that prolactin (PRL) secreted from the pituitary gland plays an important role in a variety of human immune responses. However, the immunoregulation of autocrine PRL in T lymphocytes is not fully understood. To evaluate the role of autocrine PRL in T lymphocyte activation, PRL receptor (PRLR) in Jurkat cells was silenced by lentivirus-mediated stable expression of PRLR shRNAi. Knockdown of PRLR resulted in a considerable reduction of phytohemagglutinin (PHA)-induced T cell proliferation. Moreover, the synthesis and secretion of CD137, CD154, IL-2 and IL-4 were significantly decreased, while the production of CD28, IFN-γ and IL-10 was not affected in PHA-primed PRLR-deficient cells. These results demonstrate the importance of autocrine regulation of the PRL signaling in T lymphocyte growth and activation, and support a mechanism by which autocrine PRL participates in the immunoregulation through selectively influencing the expression of certain critical costimulatory molecules and cytokines.     

Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response

Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of tumorigenicity may make AFS cells a superior source of stable, well characterized "off the shelf" immunomodulatory cells for a variety of immunotherapies.     

Listeria monocytogenes Alters Mast Cell Phenotype,Mediator and Osteopontin Secretion in a Listeriolysin-Dependent Manner

Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO) is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit) on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Inhibition of lymphocyte proliferation by antibodies to prolactin

Recent in vivo studies have shown that treatments that decrease circulating prolactin (PRL) in rodents result in significant immunosuppression. Our attempts to demonstrate corresponding direct stimulatory effects of PRL on cultured lymphocytes were unsuccessful. However, antibodies against pituitary PRL potently inhibited both murine and human lymphocyte proliferation in response to both T and B cell mitogens. Further studies using IL 2 and IL 4 responsive cell lines (CTLL-2 and HT-2) demonstrated that the same anti-PRL antibodies inhibited the proliferative response to these cytokine growth factors. Thus, antibodies to PRL appear to block an event occurring in the G1 to GS phase transition of these cell lines, which constitutively express growth factor receptors. The inhibitory activity of anti-PRL antibodies could be adsorbed by addition of purified human PRL or by immobilized PRL on an affinity column. Antibodies to other pituitary hormones were without inhibitory effect on CTLL-2 cell proliferation. Proliferation of lymphocytes in serum-free medium was also potently inhibited by anti-PRL antibodies, suggesting that antibody effects were not due to neutralization of PRL or other factors contained in culture serum supplements. We suggest from these data that a protein with homology to PRL and recognized by these anti-PRL antibodies is produced by lymphocytes and plays a critical role in their progression through the cell cycle.     

Interleukin-2 in human milk: a potential modulator of lymphocyte development in the breastfed infant

Development of lymphocyte subpopulations and response to antigen exposure will be influenced by the limited ability of neonates to produce cytokines. In the case of cytokines such as interleukin (IL)-2 which are potent T lymphocyte regulators but poorly produced by newborn infants, the supply of cytokines through human milk could alleviate an immunological deficit and potentially aid the maturation of the immune system. We analysed human milk from 52 mothers (15-357 days postpartum) by ELISA to determine levels of aqueous IL-2, as well as production by human milk cells. IL-2 was detectable (>8 pg/mL) in the aqueous phase of 81% of all day 1 samples with no significant difference found in the mean concentration over 3 consecutive days. IL-2 was produced constitutively at detectable levels by 57% of milk cell samples and production was significantly increased by stimulation with Con A (380%). No correlation was found between aqueous and cellular IL-2, however there was a significant correlation between milk aqueous IL-2 and serum IL-2. This is the first report of IL-2 in the aqueous phase of human milk. A supply of exogenous IL-2 in human milk may provide the suckling infant with important immunological signals during a significant stage of T cell development.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

Immune system development and function in prolactin receptor-deficient mice.

Prolactin (PRL) is the primary lactogenic pituitary hormone that plays an essential role in many aspects of reproduction, from fertilization to mammary gland development and maternal behavior. PRL has also been reported to play a role in immunoregulation. Because initial observations indicated that hypophysectomized rats present abnormalities of the immune system, including increased thymic atrophy and lymphopenia, a number of studies have focused on the potential immunomodulatory roles of PRL. This hormone exerts its biological activities following binding to specific cell surface PRL receptors (PRLRs). In this report, we have characterized the development and function of the immune system in PRLR-deficient mice. Compared with wild-type control mice, PRLR-/- mice demonstrate no alterations in thymic or splenic cellularity or in the composition of the lymphocyte subsets present in primary (bone marrow and thymus) or secondary (spleen and lymph nodes) lymphoid organs. Lymphocytes from PRLR-/- mice are functional in vitro, as they can proliferate normally to mitogens, cytokines, and allogeneic cells. PRLR-/- splenocytes display normal NK-mediated cytotoxicity to YAC-1 target cells. In vivo studies have revealed that PRLR-/- mice are able to 1) generate normal steady-state Ig levels, 2) mount a normal specific Ig response following immunization with a T-dependent Ag, 3) eliminate injected allogeneic tumor cells, and 4) effectively control Listeria monocytogenes infection. Taken together, these results show that immune system development and function proceed normally in the absence of PRL-mediated signaling and suggest that PRLR pathways are not essential for immunomodulation in vivo.     

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.     

Recent Advances in the Characterization of Genetic Factors Involved in Human Susceptibility to Infection by Schistosomiasis

Human resistance to infection by schistosomes is associated to a strong Th2 immune. However a persistent Th2 response can cause severe kidney and liver disease in human. In this review, we mainly focused on the control of infection levels caused by schistosomes. Several experimental models allowed us to better understand the immunological mechanisms of the host against schistosome infection. High IgE and eosinophil levels are associated with resistance to infection by schistosomes and this effect is counterbalanced by IgG4. IgE and eosinophils are highly dependent on IL-4, IL-13, and Il-5, which are three main Th2 cytokines. We also examined the genetic factors involved in human susceptibility to infection by schistosomiasis. Infection levels are mainly regulated by a major locus SM1, in 5q31-q33 region, which contains the genes encoding for the IL-4, IL-13, and Il-5 cytokines. An association between an IL13 polymorphism, rs1800925, and infection levels has been shown. This polymorphism synergistically acts with another polymorphism (rs324013) in the STAT6 gene, encoding for the signal transducer of the IL13 pathway. This pathway has also been involved in atopic disorders. As helminthiasis, atopy is the result of aberrant Th2 cytokine response to allergens, with an increased production of IL-4, IL-13, Il-9 and Il-5, with high amounts of allergen-specific and total IgE and eosinophilia. However, the Th2 immune response is protective in helminthiasis but aggravating in atopic disorders. Several studies reported interplay between helminthic infections and allergic reactions. The different results are discussed here.     

TNF and IL-10 are major factors in modulation of the phagocytic cell environment in lung and lymph node in tuberculosis: A next-generation two-compartmental model

Tuberculosis (TB) is one of the earliest recorded human diseases and still one of the deadliest worldwide. Its causative agent is the bacteria Mycobacterium tuberculosis (Mtb). Cytokine-mediated macrophage activation is a necessary step in control of bacterial growth, and early immunologic events in lymph node and lung are crucial to the outcome of infection, although the factors that influence these environments and the immune response are poorly understood.Our goal is to build the next-generation two-compartmental model of the immune response to provide a gateway to more spatial and mechanistic investigations of M. tuberculosis infection in the LN and lung. Crucial immune factors emerge that affect macrophage populations and inflammation, namely TNF-dependent recruitment and apoptosis, and IL-10 levels. Surprisingly, bacterial load plays a less important role than TNF in increasing the population of infected macrophages and inflammation.Using a mathematical model, it is possible to distinguish the effects of pro-inflammatory (TNF) and anti-inflammatory (IL-10) cytokines on the spectrum of phagocyte populations (macrophages and dendritic cells) in the lung and lymph node. Our results suggest that TNF is a major mediator of recruitment of phagocytes to the lungs. In contrast, IL-10 plays a role in balancing the dominant macrophage phenotype in LN and lung.     

Regulation of Intestinal Immune Response by Selective Removal of the Anterior,Posterior, or Entire Pituitary Gland in Trichinella spiralis Infected Golden Hamsters

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been previously reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, and reduction of gastric secretion and intestinal absorption, as well as increased susceptibility to bacterial and viral infections. However, to our knowledge, no findings have been published concerning the immune response following HYPOX during worm infection, particularly that which is caused by the nematode Trichinella spiralis. The aim of this work was to analyze the effects of total or partial HYPOX on colonization of T. spiralis in the intestinal lumen, together with duodenal and splenic cytokine expression. Our results indicate that 5 days post infection, only neurointermediate pituitary lobectomy (NIL) reduces the number of intestinally recovered T. spiralis larvae. Using semiquantitative inmunofluorescent laser confocal microscopy, we observed that the mean intensity of all tested Th1 cytokines was markedly diminished, even in the duodenum of infected controls. In contrast, a high level of expression of these cytokines was noted in the NIL infected hamsters. Likewise, a significant decrease in the fluorescence intensity of Th2 cytokines (with the exception of IL-4) was apparent in the duodenum of control and sham infected hamsters, compared to animals with NIL surgeries, which showed an increase in the expression of IL-5 and IL-13. Histology of duodenal mucosa from NIL hamsters showed an exacerbated inflammatory infiltrate located along the lamina propria, which was related to the presence of the parasite. We conclude that hormones from each pituitary lobe affect the gastrointestinal immune responses to T. spiralis through various mechanisms.     

Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Macrophages as IL-25/IL-33-Responsive Cells Play an Important Role in the Induction of Type 2 Immunity

Type 2 immunity is essential for host protection against nematode infection but is detrimental in allergic inflammation or asthma. There is a major research focus on the effector molecules and specific cell types involved in the initiation of type 2 immunity. Recent work has implicated an important role of epithelial-derived cytokines, IL-25 and IL-33, acting on innate immune cells that are believed to be the initial sources of type 2 cytokines IL-4/IL-5/IL-13. The identities of the cell types that mediate the effects of IL-25/IL-33, however, remain to be fully elucidated. In the present study, we demonstrate that macrophages as IL-25/IL-33-responsive cells play an important role in inducing type 2 immunity using both in vitro and in vivo approaches. Macrophages produced type 2 cytokines IL-5 and IL-13 in response to the stimulation of IL-25/IL-33 in vitro, or were the IL-13-producing cells in mice administrated with exogenous IL-33 or infected with Heligmosomoides bakeri. In addition, IL-33 induced alternative activation of macrophages primarily through autocrine IL-13 activating the IL-4Rα-STAT6 pathway. Moreover, depletion of macrophages attenuated the IL-25/IL-33-induced type 2 immunity in mice, while adoptive transfer of IL-33-activated macrophages into mice with a chronic Heligmosomoides bakeri infection induced worm expulsion accompanied by a potent type 2 protective immune response. Thus, macrophages represent a unique population of the innate immune cells pivotal to type 2 immunity and a potential therapeutic target in controlling type 2 immunity-mediated inflammatory pathologies.