OBJECTIVE: Recent investigations of schizophrenia have targeted glutamatergic neurotransmission, since phencyclidine, an N-methyl-d-aspartate (NMDA) receptor antagonist, can induce schizophreniform psychosis. The authors previously reported alterations in thalamic NMDA receptor subunit expression in schizophrenia, consistent with the hypothesis that thalamic glutamatergic hypofunction may contribute to the pathophysiology of this illness. In this study they generalized this hypothesis to include other molecules of the glutamate synapse, specifically excitatory amino acid transporters (EAATs), whose normal expression and regulation in the thalamus may also be disrupted in subjects with schizophrenia. METHOD: In situ hybridization with riboprobes specific for the human excitatory amino acid transporter transcripts EAAT1, EAAT2, and EAAT3 was performed in discrete thalamic nuclei in persons with schizophrenia and comparison subjects. RESULTS: Higher expressions of transcripts encoding EAAT1 and EAAT2, but not EAAT3, were detected in the thalamus of subjects with schizophrenia. CONCLUSIONS: These findings support the hypothesis of glutamatergic dysfunction in schizophrenia and suggest that molecules other than glutamate receptors are abnormally expressed in glutamatergic synapses in this illness.

Abnormalities of the ionotropic glutamate receptors (N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid [AMPA], and kainate) have been reported in the brain in schizophrenia, although in complex, region-specific patterns. While limbic cortex and medial temporal lobe structures have been most often studied in psychiatric illnesses, glutamate receptors are expressed in other brain regions associated with limbic circuitry, especially the striatum. In this study, we have determined striatal ionotropic glutamate receptor expression in brains from persons with schizophrenia, bipolar disorder, major depression, and a comparison group, using samples from the Stanley Foundation Neuropathology Consortium. We have determined the expression of these receptors at multiple levels of gene expression by using both in situ hybridization and receptor autoradiography. The expression of nearly all of these molecules was not different in these psychiatric conditions. The only significant changes noted were NR2D and gluR1 transcripts, and [3H]AMPA binding. This is the first comprehensive study of striatal ionotropic glutamate receptor expression in schizophrenia and affective disorders, and suggests that there are minimal changes in these receptors in this region of the brain in these illnesses.

The strong positive correlation between typical antipsychotic potency and dopamine D2 receptor affinity suggested that the mechanism of action of antipsychotics may be D2 receptor antagonism. The introduction of atypical antipsychotics has led to a reformulation of antipsychotic action. Atypical antipsychotics have different receptor antagonist properties, differentially affect neuroanatomical regions and circuits, and display unique therapeutic profiles. However, there remains some disagreement over what differentiates typical and atypical antipsychotics. One way to categorize antipsychotics takes advantage of all of the above properties: studies of neurochemical anatomy can potentially account for the clinical, pharmacological, and anatomical properties of antipsychotics, by examining receptor expression in motor and limbic circuits. We will review rodent studies examining the effects of antipsychotics on dopamine, serotonin, glutamate, GABA, and cholinergic receptor expression in limbic and motor circuits. These studies demonstrate that typical and atypical antipsychotics induce unique, region-specific patterns of receptor expression that may be the substrate of their beneficial clinical effects, as well as their side effects. Despite numerous studies to date, this literature is plagued with inconsistency, and it is difficult to reconcile all of the available data. Due to difficulties in making comparisons among the myriad experimental methodologies, and the pitfalls of attempting to generalize rodent data to human postmortem studies of schizophrenia, future studies on the effects of typical and atypical antipsychotics on receptor expression will need to: (1) simultaneously examine multiple neurotransmitter systems in the same sets of animals treated with typical and atypical antipsychotics; (2) examine multiple receptor subtypes of the same neurotransmitter system; (3) measure receptor expression in multiple, interconnected regions that comprise limbic and motor circuits; (4) measure receptor expression at multiple levels of gene organization.

Evidence suggests that abnormal glutamatergic transmission may be involved in the pathophysiology of schizophrenia since phencyclidine (PCP), an antagonist of the NMDA receptor, produces symptoms akin to positive and negative schizophrenic symptoms. Normal glutamatergic transmission relies on the presence of various receptors, co-factors, and other proteins such as amino acid transporters and receptor anchoring proteins. Anchoring proteins insert glutamate receptors in the synaptic membrane, facilitating receptor interactions with various intracellular components. Alterations of the NMDA receptor-anchoring protein interaction may affect normal receptor function. Specific anchoring proteins are associated with specific regions of the NMDA NR1 subunit, coded in exon 21 and/or 22. We have previously reported abnormal expression of total NR1 mRNA levels in schizophrenic thalamus. Given the relationship of anchoring proteins and the 3′ exons of this gene, we hypothesized that this abnormality may be specifically associated with exon 21 or 22 containing isoforms of the NR1 subunit. Using in situ hybridization we examined exon 22 mRNA expression in postmortem thalamic tissue from 13 schizophrenic and 9 control subjects, identifying six thalamic nuclei: anterior, dorsomedial, lateral dorsal, central medial, reticular, and nuclei of the ventral tier. Preliminary data indicate that there is diminished exon 22 mRNA expression in schizophrenic thalamus compared to controls. Subsequent studies will examine expression of exon 5 and 21 mRNA in these regions. These data reveal a reduction in exon 22 containing isoforms of the NR1 subunit in schizophrenic thalamus, and may suggest a physiological basis for diminished activity of the NMDA receptor in schizophrenia.

Despite forty years of investigation, the mechanism of action of antipsychotic medications is not well known. The initial characterization of antipsychotics revealed modulatory effects on dopaminergic pathways, but recent work has indicated effects upon multiple neurotransmitter systems. These receptor-mediated effects of antipsychotic medications converge on multiple signaling pathways. Modulation of cyclic nucleotide and phosphoinositide signaling has been directly linked to receptors targeted by antipsychotic medications. Despite the breadth of previous work, mechanistic formulation of the molecular effects of antipsychotic medications on signal transduction pathways has not been well synthesized. A review of the relevant literature for antipsychotic regulation of signaling processes will be presented. Specific differences between typical and atypical antipsychotics will be highlighted. Where relevant, molecular signaling processes will be linked to both therapeutic and adverse effects of antipsychotic medications. Finally, a model integrating different receptor families and signal transduction pathways with gene expression and clinical outcomes will be synthesized. This model will assist in the formulation of future studies examining the effects of typical and atypical antipsychotics on signaling pathways.

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis. For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis are dependent in part upon an overexpression of the angiogenic CXC chemokine, macrophage inflammatory protein-2 (MIP-2). To test this hypothesis, we measured MIP-2 by specific ELISA in whole lung homogenates in either bleomycin-treated or control CBA/J mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Neutralizing anti-MIP-2 Abs inhibited the angiogenic activity of day 16 bleomycin-treated lung specimens using an in vivo angiogenesis bioassay. Furthermore, when MIP-2 was depleted in vivo by passive immunization, bleomycin-induced pulmonary fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or collagen gene expression. This was also paralleled by a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis.

The pulmonary fibroblast within the interstitial space of the lung has assumed a vastly expanded role over the last 15 years. While this cell is still understood to supply important structural and support proteins to its environment, the fibroblast is now also viewed as an active participant in immune and inflammatory processes within the lung. This novel role for the lung fibroblast is facilitated through a number of mechanisms, including de novo expression of contractile smooth-muscle actin, adhesion molecules, and chemotactic cytokines or chemokines. Through these and possibly other processes, the lung fibroblast is able to interface uniquely with resident and infiltrating immune cells, such as macrophages and monocytes, T cells, mast cells, and eosinophils. While the precise contributions of these cell-to-cell interactions to the initiation and maintenance of diseases in the lung are unknown, modulation of these cellular events may provide an effective treatment option in many clinical pulmonary diseases.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

Although many studies have characterized soluble factors that stimulate or inhibit chemokine secretion, in this review we focus on the event of cellular adhesion as a novel mechanism for stimulating chemokine expression. Recent work has demonstrated chemokine expression following cell-to-cell and cell-to-matrix adhesion. The specificity of this finding was demonstrated utilizing various techniques that illustrate that adhesion, and not a soluble stimulus, is in some cases responsible for initiating or augmenting chemokine expression. For example, co-cultures of peripheral blood monocytes and endothelial cells secreted elevated levels of IL-8 and MCP-1 compared with either cell type alone. When co-cultured in transwells, this effect was significantly attenuated. In other experiments, neutralizing monoclonal antibodies to various adhesion molecules inhibited chemokine expression. The effects of adhesion were not limited to leukocytes. Both immune and non-immune cell types were evaluated as potential sources of adhesion-mediated chemokine expression. Not suprisingly, expression of some chemokines was associated with adhesion, whereas others were not, supporting the notion that adhesion differentially signals chemokine secretion during the inflammatory response. We hypothesize that as a recruited leukocyte encounters different adhesion substrates such as endothelial cells, basement membrane, extracellular matrix, and fibroblasts, the expression of chemokines from both the leukocyte and the substrate may be initiated, inhibited, or augmented. Careful characterization of the contribution of adhesion to regulation of chemokine expression will provide insight into the pathogenesis of many human diseases where chemokines have a central role.

In rodents, bleomycin administration results in a route-, dose- and strain-dependent pulmonary inflammatory response. Given intratracheally, this response is characterized by increases in leukocyte accumulation, fibroblast proliferation, and collagen content. We believe that characterization of the cell types and soluble mediators present in the lesion will lend significant insight into the processes modulating pulmonary fibrosis. Recent studies have identified monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) as mediators of the inflammatory response in the lungs of human patients afflicted with idiopathic pulmonary fibrosis. Based on this compelling evidence for the involvement of C-C chemokines in fibrotic pathologies, we investigated the roles of MIP-1 alpha and MCP-1 protein in bleomycin-induced lung injury. In this study, we have established that neutralization of MIP-1 alpha and MCP-1 significantly reduces inflammatory cell accumulation. Further, we have shown that passive immunotherapy with either anti-MCP-1 or anti-MIP-1 alpha antibodies significantly reduced mononuclear phagocyte accumulation in bleomycin-challenged mice. These experiments strongly support the hypothesis that MIP-1 alpha and MCP-1 contribute to the recruitment of leukocytes during the pulmonary inflammatory response to bleomycin challenge.

"C-C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge.." American journal of respiratory cell and molecular biology, 15(6), pp. 693–702

Pulmonary fibrosis is the end point of a chronic inflammatory process characterized by leukocyte recruitment and activation, fibroblast proliferation, and increased extracellular matrix production, Previous studies of models of pulmonary fibrosis have investigated the role of cytokines in the evolution of the fibrotic response, The involvement of tumor necrosis factor and interleukin-1 in bleomycin-induced lung injury, a model of idiopathic pulmonary fibrosis, has been well established, suggesting that cytokines mediate the initiation and maintenance of chronic inflammatory lesions, However, the aforementioned cytokines alone cannot account for the recruitment and activation of specific leukocyte populations found in the bleomycin model, Recently, a family of novel proinflammatory cytokines (chemokines) was cloned and characterized, yielding many putative mediators of leukocyte functions, Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemoattractant protein-1 (MCP 1) belong to the C-C chemotactic cytokine family, a group of low-molecular-weight peptides, These molecules modulate chemotaxis, proliferation, and cytokine expression in leukocyte subsets, Our group has investigated the roles of MCP-1 and MIP-1 alpha in the bleomycin model, Both MCP-1 and MIP-1 alpha are expressed in a time-dependent manner after bleomycin challenge, and passive immunization of these animals with either anti-MIP-1 alpha or anti-MCP-1 antibodies attenuated leukocyte accumulation, In addition, we have identified specific cell types expressing MCP-1 or MIP-1 alpha by in situ hybridization and immunohistochemical localization, respectively, Furthermore, our results indicate that MIP-1 alpha expression is mediated by alveolar macrophage-derived tumor necrosis factor, identifying an important cytokine pathway in the initiation of pulmonary fibrosis, Finally, anti-MIP-1 alpha therapy attenuated fibrosis, providing direct evidence for its involvement in fibrotic pathology, Our work has clearly established that the C-C chemokines MCP-1 and MIP-1 alpha are expressed and contribute to the initiation and maintenance of the bleomycin-induced pulmonary lesion.

We investigated the role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in bleomycin-induced lung injury, a model of interstitial lung disease. Bleomycin stimulates a T cell-dependent pulmonary inflammatory response characterized by an increase in leukocyte infiltration, fibroblast proliferation, and collagen synthesis. Intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time-dependent increase in MIP-1 alpha protein levels both in whole-lung homogenates and bronchoalveolar lavage fluid. The kinetics of MIP-1 alpha expression were biphasic, with the first peak occurring at 2 days postinstillation and the second peak at 16 days. These levels of Ag expression temporally correlated with the accumulation of granulocytes, lymphocytes, and mononuclear phagocytes in the lung. In addition, immunohistochemical staining identified alveolar macrophages and bronchial epithelial cells as the primary cellular sources of MIP-1 alpha production. Interestingly, passive immunization of bleomycin-challenged mice with anti-MIP-1 alpha Abs significantly reduced pulmonary mononuclear phagocyte accumulation and fibrosis. These experiments establish that MIP-1 alpha protein is expressed in the lungs of bleomycin-treated mice and provide evidence that MIP-1 alpha promotes leukocyte accumulation and activation. Furthermore, these findings support the notion that leukocyte accumulation and activation are linked to fibrosis.

The formation of hepatic granulomas around persistently deposited Schistosoma mansoni eggs leads to parenchymal damage, ongoing fibrosis, and ultimate loss of liver function. In this study, the production of macrophage inflammatory protein-1 alpha (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) by granuloma fibroblasts was examined to establish the potential contribution of intragranuloma fibroblasts to the maintenance of the chronic inflammation. Isolated fibroblasts from dispersed acute infection hepatic granulomas were grown in tissue culture for 3 to 4 weeks and used on the third or fourth passage. We initially surveyed fibroblasts for production of MIP-1 and MCP-1 by reverse transcription-polymerase chain reaction (RT-PCR) after stimulation with interleukin (IL)-1, tumor necrosis factor, interferon (IFN)-gamma, IL-4, or IL-10: cytokines found within the granuloma. These studies demonstrated constitutive expression of MCP-1 and differential up-regulation of MIP-1 on cytokine stimulation. Protein expression was then verified by immunohistochemical localization of MIP-1 and MCP-1 in paraformaldehyde-fixed fibroblasts and by direct quantitation of MIP-1 and MCP-1 in culture supernatants by specific ELISAs. These studies demonstrated constitutive expression of MCP-1 in unstimulated and cytokine-stimulated granuloma fibroblasts. In contrast, IL-1 (0.1 to 2.5 ng/ml), IFN-gamma (10 micrograms/ml), and IL-10 (2.5 to 10 ng/ml) were able to induce the significant production of MIP-1 by the granuloma fibroblasts. Interestingly, normal noninflammatory fibroblasts from uninfected mice showed no significant production of MIP-1 or MCP-1 in response to these cytokines. These results suggest that granuloma fibroblasts may be phenotypically altered compared with normal fibroblasts and have a significant role in leukocyte recruitment, granuloma growth, and maintenance of the egg-induced lesion.