Differential expression of the isoforms for the monocyte chemoattractant protein-1 receptor, CCR2, in monocytes.

Two isoforms of human CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), have been identified but their relative expression in monocytes and contribution to inflammatory responses mediated by MCP-1 remain uncertain. All available information on CCR2 expression is based on mRNA data because isoform-specific antibodies were not available until now. To analyze the relative expression of each isoform, we made two antibodies that specifically recognized CCR2A and CCR2B. Examination of receptor protein with these isoform-specific antibodies showed that the total expression of CCR2B in monocytes was about 10-fold higher than that of CCR2A with an equal distribution between the cell surface and intracellular pools. A detailed analysis using purified plasma membranes demonstrated that about 90% of all CCR2 on the cell surface were composed of CCR2B. The relatively abundant expression of CCR2B on the cell surface suggests a principal role of this isoform as a mediator of monocyte responses to MCP-1 in inflammation.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

Activation of PKC-beta isoforms mediates HNE-induced MCP-1 release by macrophages

4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 microM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of beta-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the delta isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.     

Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

Modulatory effect of HHV-6 on MCP-1 production by human monocytes

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.     

Spastin Couples Microtubule Severing to Membrane Traffic in Completion of Cytokinesis and Secretion

Mutations in the gene encoding the microtubule (MT)-severing protein spastin are the most common cause of hereditary spastic paraplegia, a genetic condition in which axons of the corticospinal tracts degenerate. We show that not only does endogenous spastin colocalize with MTs, but that it is also located on the early secretory pathway, can be recruited to endosomes and is present in the cytokinetic midbody. Spastin has two main isoforms, a 68 kD full-length isoform and a 60 kD short form. These two isoforms preferentially localize to different membrane traffic pathways with 68 kD spastin being principally located at the early secretory pathway, where it regulates endoplasmic reticulum-to-Golgi traffic. Sixty kiloDalton spastin is the major form recruited to endosomes and is also present in the midbody, where its localization requires the endosomal sorting complex required for transport-III-interacting MIT domain. Loss of midbody MTs accompanies the abscission stage of cytokinesis. In cells lacking spastin, a MT disruption event that normally accompanies abscission does not occur and abscission fails. We suggest that this event represents spastin-mediated MT severing. Our results support a model in which membrane traffic and MT regulation are coupled through spastin. This model is relevant in the axon, where there also is co-ordinated MT regulation and membrane traffic.     

Borrelia burgdorferi-induced monocyte chemoattractant protein-1 production in vivo and in vitro

Matrix metalloproteinase 9 (MMP-9) is selectively upregulated in erythema migrans (EM) lesions with acute Lyme disease. This study explored whether upregulation of MMP-9 was associated with monocyte chemoattractant protein 1 (MCP-1) production, and Borrelia burgdorferi (B. burgdorferi) could induce MCP-1 production in vivo and in vitro. The results indicated that expression of MCP-1 was significantly increased in U937 cells by B. burgdorferi. The activity of MMP-9 could be elevated by recombinant MCP-1 (rMCP-1) in U937 cells. MMP-9 was not upregulated by B. burgdorferi in fibroblasts. However, the expression of MCP-1 was significantly increased in the presence of B. burgdorferi in fibroblasts. The level of MCP-1 in EM lesions and in serum of patients with acute Lyme disease was also significantly elevated compared to that for healthy controls. The secreted MCP-1 may affect the production of MMP-9 in fibroblasts and/or macrophages.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.     

MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma

Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis.     

Monocyte chemoattractant protein-1 selectively inhibits the acquisition of CD40 ligand-dependent IL-12-producing capacity of monocyte-derived dendritic cells and modulates Th1 immune response

Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, also displays immunoregulatory functions and may be involved in Th subset differentiation. In this study, we examined the effects of MCP-1 on the cytokine-driven differentiation of monocytes into dendritic cells (DCs), the most potent APCs for naive T cells. We found that DCs generated in the presence of MCP-1 displayed a markedly reduced production of IL-12 in response to CD40 ligand but not in response to Staphylococcus aureus stimulation in the presence or absence of IFN-gamma. The production of IL-10, a potent endogenous IL-12 inhibitor, was not affected by MCP-1. Whereas the inhibitory activity of MCP-1 on IL-12 production by monocytes was sensitive to pertussis toxin, its effects on DC differentiation were pertussis toxin resistant. MCP-1 did not affect the surface phenotype and T cell-stimulating activity of DCs, but most interestingly, naive T cells stimulated with MCP-1-primed DCs produced much less IFN-gamma but the same levels of IL-13. Taken together, our results indicated that MCP-1 modulates the differentiation of monocytes into DCs and may thereby inhibit Th1 cell development.     

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.     

Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2

The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.     

Role of glucocorticoids and Toxoplasma gondii infection on murine intestinal epithelial cells

Glucocorticoids (GCs) are stress hormones secreted in response to perceived psychological and or physiological stress. GCs have been shown to reduce tissue inflammation by down-regulating the production of inflammatory chemokines produced by epithelial cells. The protozoan parasite Toxoplasma gondii is known to increase cytokine, chemokine, and Toll-like receptors (TLRs) expression in parasite infected mouse intestinal epithelial cells (IECs). We sought to analyze the role of an anti-inflammatory protein, glucocorticoid-induced leucine zipper (GILZ) in MODE-K cells during infection with T. gondii. GILZ expression in MODE-K cells was assessed by PCR and immunoblotting after stimulation with GCs (corticosterone, CORT) or T. gondii infection. GILZ mRNA was constitutively expressed in MODE-K cells but not its protein product. While infection and pre-exposure to CORT decreased GILZ isoforms of 28 and 17 kD, the presence of CORT during infection increased levels of 17 kD isoform. Infected cells treated with CORT had decreased expression of chemokines (IP-10/CXCL10, MCP-1/CCL2, MIP-2/CXCL8) while their expression was increased when endogenous GILZ was removed by siRNA treatment. GILZ up-regulation during infection may serve as a mechanism to decrease epithelial cell responses and facilitate parasite replication.     

Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice

Intratracheal instillation of the monocyte chemoattractant JE/monocyte chemoattractant protein (MCP)-1 in mice was recently shown to cause increased alveolar monocyte accumulation in the absence of lung inflammation, whereas combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute lung inflammation with early alveolar neutrophil and delayed alveolar monocyte influx. We evaluated the role of resident alveolar macrophages (rAM) in these leukocyte recruitment events and related phenomena of lung inflammation. Depletion of rAM by pretreatment of mice with liposomal clodronate did not affect the JE/MCP-1-driven alveolar monocyte accumulation, despite the observation that rAM constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by flow cytometry and immunohistochemistry. In contrast, depletion of rAM largely suppressed alveolar cytokine release as well as neutrophil and monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment. Despite this strongly attenuated alveolar inflammatory response, increased lung permeability was still observed in rAM-depleted mice undergoing JE/MCP-1/LPS challenge. Lung leakage was abrogated by codepletion of circulating neutrophils or administration of anti-CD18. Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs but largely contribute to the alveolar cytokine response and enhanced early neutrophil and delayed monocyte influx under inflammatory conditions (JE/MCP-1/LPS deposition). Loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils via beta(2)-integrin engagement.     

Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon

Bicarbonate and butyrate stimulate electroneutral Na absorption via apical membrane Na-H exchange (NHE) in rat distal colon. cAMP downregulates NHE-3 isoform and inhibits HCO3-dependent, but not butyrate-dependent, Na absorption. This study sought to determine whether 1) the apical membrane NHE-2 and NHE-3 isoforms differentially mediated HCO3- and butyrate-dependent Na absorption, and 2) cAMP had different effects on NHE-2 and NHE-3 isoforms. The effect of specific inhibitors of NHE-2 and NHE-3 isoforms (50 microM HOE 694 and 2 microM S3226, respectively) on unidirectional 22Na transepithelial fluxes performed across isolated mucosa from rat distal colon under voltage-clamp conditions was examined. HCO3 stimulation of Na absorption was inhibited by EIPA, a nonspecific inhibitor of all NHE isoforms, by S3226 and dibutyryl cAMP but not by HOE 694. In contrast, butyrate stimulation of Na absorption was not altered by dibutyryl cAMP and was not inhibited by HOE 694 in the absence of dibutyryl cAMP, but in the presence of dibutyryl cAMP was HOE694 sensitive. In contrast, S3226 inhibited butyrate-stimulated Na absorption in the absence of dibutyryl cAMP, but not in its presence. We conclude that 1) HCO3-stimulated Na absorption is mediated solely by NHE-3 isoform, whereas butyrate-stimulated Na absorption is mediated by either NHE-3 or NHE-2 isoform, and 2) dibutyryl cAMP selectively inhibits NHE-3 isoform but stimulates NHE-2 isoform. Dibutyryl cAMP does not inhibit butyrate-stimulated Na absorption as a result of its differential effects on NHE-2 and NHE-3 isoforms.     

Overexpression of acyl carrier protein-1 alters fatty acid composition of leaf tissue in Arabidopsis

Acyl carrier protein (ACP) is a small (9 kD) acidic protein that is an essential cofactor in plant fatty acid biosynthesis. Most plants have several isoforms of ACP, some of which are expressed constitutively and others that appear to be more tissue specific. Although the critical role of ACP in fatty acid biosynthesis has been established, the role of the diverse number of isoforms has yet to be elucidated. We have generated transgenic Arabidopsis plants that express high levels of ACP-1, a seed-predominant ACP isoform, in leaf tissue under control of the cauliflower mosaic virus 35S promoter. Western and northern analysis of these plants demonstrate 3- to 8-fold increased expression of this isoform in leaf tissue, but no significant changes in seed. Analysis of the fatty acid composition of leaf tissue revealed that overexpression of ACP-1 in leaf tissue alters fatty acid composition. Significant decreases in levels of 16:3 were noted along with increases in 18:3. These findings represent the first in vivo report that overexpression of an ACP isoform results in changes in fatty acid composition in plants.