Neuroprotective role of the innate immune system by microglia

Innate immunity is a rapid series of reactions to pathogens, cell injuries and toxic proteins. A key component of this natural response is the production of inflammatory mediators by resident microglia and infiltrating macrophages. There is accumulating evidence that inflammation contributes to acute injuries and more chronic CNS diseases, though other studies have shown that inhibition of microglia is, in contrast, associated with more damages or less repair. The controversies regarding the neuroprotective and neurodegenerative properties of microglia may depend on the experimental approaches. Neurotoxic substances are frequently used to produce animal models of acute injuries or diseases and they may activate microglia either directly or indirectly by their ability to cause neuronal death and demyelination. Whether microglia and the immune response play a direct role in such processes still remains an open question. On the other hand, there are data supporting the role of resident microglia and those derived from the bone marrow in the stimulation of myelin repair, removal of toxic proteins from the CNS and the prevention of neurodegeneration in chronic brain diseases. The ability of glucocorticoids to provide a negative feedback on nuclear factor kappa B pathways in microglia may be a determinant mechanism underlying the ultimate fate of the inflammatory response in the CNS. This review presents new concepts regarding the neuroprotective role of the innate immune response in the brain and how microglia can be directed to improve recovery after injuries and prevent/delay neurodegeneration.     

Coupling of presynaptic muscarinic autoreceptors to serine kinases in low and high release conditions on the rat motor nerve terminal

We used intracellular recording to investigate how muscarinic acetylcholine receptors and the serine kinase signal transduction cascade are involved in regulating transmitter release in the neuromuscular synapses of the levator auris longus muscle from adult rats. Experiments with M1 and M2 selective blockers show that these subtypes of muscarinic receptors were involved in enhancing and inhibiting acetylcholine (ACh) release, respectively. Because the unselective muscarinic blocker atropine considerably increased release, the overall presynaptic muscarinic mechanism seemed to moderate ACh secretion in normal conditions. This muscarinic function did not change when more ACh was released (high external Ca2+) or when there was more ACh in the cleft (fasciculin II). However, when release was low (high external Mg2+ or low external Ca2+) or when there was less ACh in the cleft (when acetylcholinesterase was added, AChE), the response of M1 and M2 receptors to endogenously released ACh shifted to optimize release, thus producing a net potentiation of the Mg2+-depressed level. Protein kinase A (PKA) (but not protein kinase C, PKC) has a constitutive role in promoting a component of normal release because when it is inhibited with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2 HCl, release diminishes. The imbalance of the muscarinic acetylcholine receptors (mAChRs) (with the selective block of M1 or M2) inverts the kinase function. PKC can then tonically stimulate transmitter release, whereas PKA is uncoupled. The muscarinic function can be explained by an increased M1-mediated PKC activity-dependent release and a decreased M2-mediated PKA activity-dependent release. In the presence of high external Mg2+ or low Ca2+, or when AChE is added, both mAChRs may potentiate release through an M2-mediated PKC mechanism and an M1-mediated mechanism downstream of the PKC.     

Morphology and physiology of lamina I neurons of the caudal part of the trigeminal nucleus

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin-streptavidin-Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10-90% rise time of 0.50+/-0.06 ms and a decay time constant of 2.5+/-0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10-90% rise time was 8+/-2 ms and the deactivation time constants were 94+/-31 and 339+/-72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.     

Protracted effects of chronic oral haloperidol and risperidone on nerve growth factor, cholinergic neurons, and spatial reference learning in rats

The primary therapeutic agents used for schizophrenia, antipsychotic drugs, ameliorate psychotic symptoms; however, their chronic effects on cognition (or the physiologic processes of the brain that support cognition) are largely unknown. The purpose of this rodent study was to extend our previous work on this subject by investigating persistent effects (i.e. during a 14 day drug-free washout period) of chronic treatment (i.e. ranging from 45 days to 6 months) with a representative first and second generation antipsychotic. Drug effects on learning and memory and important neurobiological substrates of memory, the neurotrophin, nerve growth factor (NGF) and its receptors, and certain components of the basal forebrain cholinergic system were investigated. Behavioral effects of oral haloperidol (2.0 mg/kg/day), or risperidone (2.5 mg/kg/day) were assessed in an open field, a water maze task, and a radial arm maze procedure and neurochemical effects in brain tissue were subsequently measured by enzyme-linked immunosorbent assays (ELISAs). The results indicated that both antipsychotics produced time-dependent and protracted deficits in the performance of a water maze procedure when compared with vehicle-treated controls, while neither drug was associated with significant alterations in radial arm maze performance. Interestingly, haloperidol, but not risperidone, was detectible in the rodent brain in appreciable levels for up to 2 weeks after drug discontinuation. Both antipsychotics were also associated with reduced levels of NGF protein in the basal forebrain and prefrontal cortex and significant (or nearly significant) decreases in phosphorylated tropomyosin-receptor kinase A (TrkA) protein and the vesicular acetylcholine transporter (depending on the brain region analyzed). Neither antipsychotic markedly affected TrkA or p75 neurotrophin receptor levels. These data in rats indicate that chronic treatment with either haloperidol or risperidone may be associated with protracted negative effects on cognitive function as well as important neurotrophin and neurotransmitter pathways that support cognition.     

Selective activation of 5-HT(2C) receptors stimulates GABA-ergic function in the rat substantia nigra pars reticulata: a combined in vivo electrophysiological and neurochemical study

In vivo electrophysiology and microdialysis were used to investigate the physiological role of 5-HT(2C) receptors in the control of substantia nigra pars reticulata (SNr) function. Extracellular single-unit recordings were performed from putative GABA-containing neurons in the SNr of anesthetized rats, and local GABA release was studied by in vivo microdialysis in the SNr of awake freely-moving rats. Systemic administration of the selective 5-HT(2C) receptor agonist (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C(4)H(4)O(4) (RO 60-0175) caused a dose-dependent excitation of about 30% of the SNr neurons recorded. However, the remaining neurons were either inhibited or unaffected by systemic RO 60-0175, in similar proportion. Local application of RO 60-0175 by microiontophoresis caused excitation in the majority of SNr neurons tested (48%), whereas a group of neurons was inhibited (16%) or unaffected (36%). Both the excitatory and the inhibitory effects of systemic and microiontophoretic RO 60-0175 were completely prevented by pretreatment with SB 243213 [5-methyl-1-({2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl}carbamoyl)-6-trifluoromethylindoline], a selective and potent 5-HT(2C) receptor antagonist. Consistent with these electrophysiological data, both systemic and intranigral administration of RO 60-0175 and m-chlorophenylpiperazine (mCPP), a non-selective 5-HT(2C) agonist, markedly increased extracellular GABA levels in the SNr. The stimulatory effect of systemic and local RO 60-0175 on GABA release was completely prevented by systemic administration of SB 243213, whereas local application of SB 243213 into the SNr only partially blocked RO 60-0175-induced GABA release. It is concluded that selective activation of 5-HT(2C) receptors stimulates GABA-ergic function in the SNr, and the clinical relevance of these data is discussed.     

A built-in co-carcinogenic effect due to viruses involved in latent or persistent infections

A new hypothesis for some cancers, which combines the chromosomal instability theories with a co-carcinogenic effect of viruses causing latent or persistent infection, is presented. The hypothesis incorporates the multi-step model of cancer and that pre-cancerous cells reach a state of chromosomal instability. Because of chromosomal instability, the genome of these cell lines will lead to changes from generation to generation and will face a remarkable selection pressure both from lost traits, apoptosis, and from the immune system. Viruses causing latent or persistent infections have evolved many different genes capable to evade the immune system. If these viruses are harboured in the genome of pre-cancerous cells they could provide them with "superpowers" and with genes that may assist the cells to elude the immune system. The theory explains why cancer predominantly is a disease of old age. Upon aging, the immune system becomes reduced including the ability to control and suppress the viruses that cause latent or persistent infections. The risk of cancer could thereby increase as the immune functions decrease. The theory provides new insights to the genesis of cancers.     

Identification of estrogen-responsive genes involved in breast cancer metastases to the bone

Bone metastasis is the most common metastasis in breast cancer patients. Clinical observations propose strong association between estrogen receptor (ER)-positive tumors and the development of bone metastases. We hypothesized of biologically diverse sets of hormone-dependent tumors predisposed to bone metastases and of possible role of ER-signaling pathways in the development and progression of bone metastases. We developed a novel in vitro estrogen (E2)-responsive model system, in which breast cancer cells and bone cells express high levels of either ERα or ERβ. Using co-culture approach and gene array technology we identified E2-responsive genes involved in the interaction between cancer cells and bone cells. We detected 13 genes that were altered solely by ERα and 11 genes that were regulated solely by ERβ in cancer cells. Only 5 genes were modified by both ERα and ERβ. Interestingly, the majority of genes in bone cells were altered through ERβ. Two genes, namely MacMarcks and Muc-1, whose changes in expressions in cancer cells in response to E2 were highly significant, were selected for immunohistochemical analysis using tissue microarrays of 59 infiltrating ductal carcinomas. Our results indicated that both MacMarcks and Muc-1 were expressed at high frequency in ER-positive tumors. The correlation between ERα- and ERβ-status of hormone-dependent tumors with combined expression of these two markers might suggest a more aggressive tumor phenotype associated with bone metastases. Further analysis of tissues with clinicopathological characteristics and known bone metastatic disease will indicate potential prognostic values of these and other markers in the development of bone metastases in a subgroup of “bad” hormone-dependent breast cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell–cell adhesion of colorectal cancer cells

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-α) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-α and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.     

Cyclooxygenase enzyme expression and E series prostaglandin receptor signalling are enhanced in heavy menstruation

BACKGROUND: Although the mechanisms underlying the causes of heavy menstrualblood loss (MBL) remain to be elucidated, prostaglandins havebeen previously implicated. This study was initiated to elucidatea pattern of expression of the various components of the cyclooxygenase(COX)–prostaglandin signalling pathways present in theendometrium of women with normal and heavy MBLs. METHODS: Endometrial biopsies were collected at different stages of themenstrual cycle from women who underwent measurement of MBL.Tissue was divided for either examination of gene expressionby quantitative RT–PCR analysis or in vitro culture experimentation. RESULTS: Analysis of gene expression demonstrated a significant elevationin expression of COX-1 and COX-2 mRNA in endometrium obtainedfrom women with heavy MBL when compared with endometrium obtainedfrom women with normal MBL. Tissue culture with PGE₂ stimulationcaused a significantly elevated production of cyclic AMP (cAMP)by endometrium of women with heavy MBL when compared with normalMBL. Expression of phosphodiesterase 4B, an enzyme involvedin cAMP breakdown, was reduced in these same endometrial samplesobtained from women with heavy MBL. CONCLUSIONS: These data identify the E series prostaglandin receptors andtheir signalling pathways as potential therapeutic targets inthe treatment of heavy menstruation.     

Mast cells in allergy: innate instructors of adaptive responses

The function of mast cells as effector cells in allergy has been extensively studied. However, increasing insight into mast cell physiology has revealed new mast cell functions and has introduced mast cells as key players in the regulation of innate as well as adaptive immunity. For example, mast cells have recently been found to express Toll-like receptors (TLRs), which enable them to participate in the innate immune response against pathogens. Furthermore, mast cells have been reported to interact with B cells, dendritic cells and T cells and thereby modulate the direction of an adaptive immune response. Finally, recent documentation that mast cells express functional MHC class II and costimulatory molecules and release immunologically active exosomes, has raised the possibility that mast cells also engage in (as yet) poorly understood antigen presentation functions. In this review, we explore the hypothesis that mast cells serve as central mediators between innate and adaptive immunity, rather as pure effector cells, during allergic innate responses.