Spinal cord vascular degeneration impairs duloxetine penetration.

Introduction: Chronic pain is a prevalent physically debilitating health-related morbidity. Frontline analgesics are inadequate, providing only partial pain relief in only a proportion of the patient cohort. Here, we explore whether alterations in spinal cord vascular perfusion are a factor in reducing the analgesic capability of the noradrenaline reuptake inhibitor, duloxetine. Method: An established rodent model of spinal cord vascular degeneration was used. Endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse was induced via hydroxytamoxifen administered via intrathecal injection. Duloxetine was administered via intraperitoneal injection, and nociceptive behavioural testing was performed in both WT and VEGFR2KO mice. LC-MS/MS was performed to explore the accumulation of duloxetine in the spinal cord in WT and VEGFR2KO mice. Results: Spinal cord vascular degeneration leads to heat hypersensitivity and a decline in capillary perfusion. The integrity of noradrenergic projections (dopa - hydroxylase labelled) in the dorsal horn remained unaltered in WT and VEGFR2KO mice. There was an association between dorsal horn blood flow with the abundance of accumulated duloxetine in the spinal cord and analgesic capacity. In VEGFR2KO mice, the abundance of duloxetine in the lumbar spinal cord was reduced and was correlated with reduced anti-nociceptive capability of duloxetine. Discussion: Here, we show that an impaired vascular network in the spinal cord impairs the anti-nociceptive action of duloxetine. This highlights that the spinal cord vascular network is crucial to maintaining the efficacy of analgesics to provide pain relief.

Diabetes-induced microvascular complications at the level of the spinal cord: a contributing factor in diabetic neuropathic pain.

KEY POINTS: Diabetes is thought to induce neuropathic pain through activation of dorsal horn sensory neurons in the spinal cord. Here we explore the impact of hyperglycaemia on the blood supply supporting the spinal cord and chronic pain development. In streptozotocin-induced diabetic rats, neuropathic pain is accompanied by a decline in microvascular integrity in the dorsal horn. Hyperglycaemia-induced degeneration of the endothelium in the dorsal horn was associated with a loss in vascular endothelial growth factor (VEGF)-A165 b expression. VEGF-A165 b treatment prevented diabetic neuropathic pain and degeneration of the endothelium in the spinal cord. Using an endothelial-specific VEGFR2 knockout transgenic mouse model, the loss of endothelial VEGFR2 signalling led to a decline in vascular integrity in the dorsal horn and the development of hyperalgesia in VEGFR2 knockout mice. This highlights that vascular degeneration in the spinal cord could be a previously unidentified factor in the development of diabetic neuropathic pain. ABSTRACT: Abnormalities of neurovascular interactions within the CNS of diabetic patients is associated with the onset of many neurological disease states. However, to date, the link between the neurovascular network within the spinal cord and regulation of nociception has not been investigated despite neuropathic pain being common in diabetes. We hypothesised that hyperglycaemia-induced endothelial degeneration in the spinal cord, due to suppression of vascular endothelial growth factor (VEGF)-A/VEGFR2 signalling, induces diabetic neuropathic pain. Nociceptive pain behaviour was investigated in a chemically induced model of type 1 diabetes (streptozotocin induced, insulin supplemented; either vehicle or VEGF-A165 b treated) and an inducible endothelial knockdown of VEGFR2 (tamoxifen induced). Diabetic animals developed mechanical allodynia and heat hyperalgesia. This was associated with a reduction in the number of blood vessels and reduction in Evans blue extravasation in the lumbar spinal cord of diabetic animals versus age-matched controls. Endothelial markers occludin, CD31 and VE-cadherin were downregulated in the spinal cord of the diabetic group versus controls, and there was a concurrent reduction of VEGF-A165 b expression. In diabetic animals, VEGF-A165 b treatment (biweekly i.p., 20 ng g-1 ) restored normal Evans blue extravasation and prevented vascular degeneration, diabetes-induced central neuron activation and neuropathic pain. Inducible knockdown of VEGFR2 (tamoxifen treated Tie2CreERT2 -vegfr2flfl mice) led to a reduction in blood vessel network volume in the lumbar spinal cord and development of heat hyperalgesia. These findings indicate that hyperglycaemia leads to a reduction in the VEGF-A/VEGFR2 signalling cascade, resulting in endothelial dysfunction in the spinal cord, which could be an undiscovered contributing factor to diabetic neuropathic pain.

A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype.

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

Involvement of retinoic acid-inducible gene-I in inflammation of rheumatoid fibroblast-like synoviocytes.

Interferon (IFN)-gamma is a major cytokine that regulates T helper 1-type immune reactions and serves as an important mediator in the pathogenesis of autoimmune diseases. Retinoic acid-inducible gene-I (RIG-I) is an IFN-gamma-inducible gene and known to be involved in the inflammatory and immune reactions. In the present study, we found high levels of RIG-I expression in synovial tissues of rheumatoid arthritis (RA), while the expression in osteoarthritis tissues was low. Treatment of cultured fibroblast-like synoviocytes with IFN-gamma markedly induced the expression of RIG-I. Knockdown of RIG-I in fibroblast-like synoviocytes, with specific siRNA, resulted in the inhibition of the IFN-gamma-induced expression of chemokine (C-X-C motif) ligand 10 (CXCL10)/IFN-gamma-inducible protein-10 (IP-10), a chemokine with chemotactic activity towards T cells. These findings suggest that RIG-I may play an important role in the pathogenesis of synovial inflammation in RA, at least in part, by regulating the IFN-gamma-induced expression of CXCL10/IP-10.

An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer.

Inflammation is a critical process for eliminating pathogens, but can lead to serious deleterious effects if left unchecked. Identifying the endogenous factors that control immune tolerance and inflammation is a key goal in the field of immunology. Galectins, a family of endogenous lectins with affinity for beta-galactoside-containing oligosaccharides, are expressed by several cells of the immune system and tissue-resident stromal cells. According to their architecture, this family of glycan-binding proteins is classified in those containing one-carbohydrate-recognition domain (CRD) (proto-type), those containing two-CRD joined by a linker non-lectin domain (tandem-repeat) and those that have one-CRD attached to an N-terminal peptide (chimera-type). Accumulating evidence indicates that galectins play critical regulatory roles in immune cell response and homeostasis. In this review, we summarize recent developments in our understanding of the galectins' roles within different immune cell compartments, and in the broader context of the inflammatory microenvironments. In particular we illustrate the immunoregulatory role of three representative members of each galectin subfamily: galectin-1, -3 and -9. This body of knowledge, documenting the coming of age of galectins as potential immunosuppressive agents or targets for anti-inflammatory drugs, represents a sound basis to further explore their potential as novel therapies for autoimmune diseases, chronic inflammation and cancer.

Regulatory mechanisms of galectin-9 and eotaxin-3 synthesis in epidermal keratinocytes: possible involvement of galectin-9 in dermal eosinophilia of Th1-polarized skin inflammation.

BACKGROUND: Skin eosinophilia is a common feature of allergic skin diseases, but it is unclear how epidermal and dermal eosinophil infiltration is controlled. To investigate regulation of localization of eosinophils in skin, we examined the regulatory mechanisms of expression of eosinophil-specific chemoattractants in dermal fibroblasts and epidermal keratinocytes. METHODS: We analyzed production of eotaxin, eotaxin-2, eotaxin-3 and galectin-9 by dermal fibroblasts and epidermal keratinocytes in response to several stimuli in vitro. RESULTS: Dermal fibroblasts produced eotaxin and eotaxin-3 in response to stimulation by interleukin (IL)-4 and/or tumor necrosis factor-alpha. Similarly, IL-4 stimulated epidermal keratinocytes to secrete eotaxin-3. However, we did not detect eotaxin mRNA expression or protein secretion by keratinocytes stimulated in vitro. Interferon (IFN)-gamma induced galectin-9 expression on dermal fibroblasts. Conversely, expression of galectin-9 on epidermal keratinocytes was dose-dependently inhibited by IFN-gamma. The immunohistochemical assays revealed that dermal fibroblasts (but not epidermal keratinocytes) in the lesional skin of psoriasis vulgaris (a Th1-polarized disease) express significant levels of galectin-9. CONCLUSION: Eotaxin-3 contributes to dermal and epidermal eosinophil infiltration in Th2-polarized skin inflammation in which IL-4 is produced. In contrast, IFN-gamma-dominated inflammation appears to mediate eosinophil extravasation into the dermis and eosinophil adhesion to dermal fibroblasts via galectin-9 in association with decreased chemoattractant activity of epidermal galectin-9. The present results reveal a novel mechanism of dermal eosinophilia in IFN-gamma-mediated skin inflammation, and reflect concerted chemoattractant production involving dermal and/or epidermal eosinophilia during changes in the local cytokine profile.

The effect of methotrexate (MTX) on expression of signalling lymphocytic activation molecule (SLAM) in patients with rheumatoid arthritis (RA) and its role in the regulation of cytokine production.

OBJECTIVE: To investigate the effect of methotrexate (MTX) on cytokine production by activated CD4+ T-cells in patients with rheumatoid arthritis (RA). METHODS: The effect of MTX on intracellular expression of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), and cell surface expression of signalling lymphocytic activation molecule (SLAM) from freshly isolated peripheral blood mononuclear cells (PBMCs), and after in vitro culture with or without MTX, was analysed with flow cytometry in 18 patients with RA and 20 healthy controls. RESULTS: Intracellular expression of IFN-gamma and IL-4 on freshly isolated CD4+ T-cells was significantly higher in patients with RA than in the controls (p<0.05). Intracellular expression of both IFN-gamma and IL-4 after culture with MTX was significantly lower than those after culture without MTX in patients with RA. Although no significant difference was observed in SLAM expression on freshly isolated CD4+ T-cells between patients with RA and the controls, MTX significantly decreased SLAM expression on both activated IFN-gamma+ and IL-4+CD4+ T-cells in patients with RA. CONCLUSION: In vitro modulation of the cytokine network by MTX, IFN-gamma, and IL-4 is one of the major targets for MTX, and production of IFN-gamma and IL-4 by PBMCs may be suppressed by SLAM on activated CD4+ T-cell in patients with RA.

Retinoic acid-inducible gene-I is induced by interleukin-1beta in cultured human gingival fibroblasts.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family protein, and details of its biological function are not known. We have studied the mechanism of the interleukin-1beta (IL-1beta)-induced RIG-I expression in human gingival fibroblasts in culture. We also addressed the possibility of enhanced expression of COX-2, RANTES and galectin-9 in fibroblasts overexpressed RIG-I. We stimulated cultured human gingival fibroblasts with IL-1beta and examined the expression of RIG-I mRNA and protein by reverse transcriptase-mediated polymerase chain reaction and Western blot analysis. The effect of cycloheximide, a protein synthesis inhibitor, on the IL-1beta-induced expression of RIG-I was examined. The expression of COX-2, RANTES, galectin-9 and monocyte chemoattractant protein-1 in gingival fibroblasts transfected with RIG-I cDNA was also examined. IL-1beta stimulated the expressions of mRNA and protein for RIG-I, in cultured fibroblasts, in a time- and concentration-dependent manner. Cycloheximide did not suppress the IL-1beta-induced RIG-I expression. Introduction of RIG-I cDNA into fibroblasts resulted in enhanced expression of COX-2 mRNA, and slightly enhanced the expression of mRNA for RANTES and galectin-9. In contrast, RIG-I overexpression did not alter the level of mRNA for monocyte chemoattractant protein-1. We conclude that IL-1beta stimulates RIG-I expression in human gingival fibroblasts.

Expression and activity analyses of CTLA4 in peripheral blood lymphocytes in systemic lupus erythematosus patients.

The objective of this study was to determine the expression and activity of CTLA4 in T-cells of systemic lupus erythematosus (SLE) patients. Expression of CTLA4 on freshly isolated peripheral blood T-cells was evaluated in 33 SLE patients and 25 controls using flow cytometry. The T-cells from 19 SLE patients and 22 controls were stimulated and cultured with Chinese hamster ovary cells expressing CD80 (CHO-CD80) or with CHO cells. T-cell proliferation was determined with [3H] thymidine incorporation (CPM), and the inhibitory effect of CTLA4 on T-cell proliferation was evaluated by the ratio of CPM for T-cells with CHO-CD80 cells to that of T-cells with CHO cells (the CHO-CD80/CHO ratio). Intracellular CTLA4 expression in freshly isolated peripheral blood T-cells was significantly higher in SLE patients than the controls (P < 0.05), but there was no correlation with clinical features or disease activity. The CHO-CD80/CHO ratio of SLE patients was significantly higher than that of the controls (P < 0.05). Among SLE patients, the CHO-CD80/CHO ratio of patients with lupus nephritis was significantly higher than that of patients without lupus nephritis (P < 0.05). In conclusion, our data suggest that CTLA4 expression is not impaired in SLE patients, but there is a possibility of decreased inhibitory effect of CTLA4 involved in the pathogenesis of SLE.

Long term prognosis of children born to lupus patients.

OBJECTIVE: To determine the long term prognosis of children of patients with systemic lupus erythematosus (SLE). METHODS: Children of patients with SLE were invited to attend our clinic for physical examination and laboratory tests. A total of 195 children (aged 4 months to 26 years; male = 82, female = 113) were examined in 1991, 1995, 1997, and 1998. RESULTS: Two cases were diagnosed as SLE at the first visit and were excluded from the second visit. A significantly higher percentage (52/195 (27%)) of patients were positive for antinuclear antibodies (ANA) at a cut off serum dilution of 1/40 compared with controls (4/57 (7%)). ANA were detected more frequently in female subjects than in men (p<0.05). Forty four subjects were examined on more than two occasions. Nine of the 10 patients who were positive for ANA at the second visit were girls aged 4-8 years. The incidence of anti-DNA and antiphospholipid antibodies in children of patients with SLE was similar to that in the controls. CONCLUSIONS: The finding that children, especially girls, born to maternal lupus patients had a high positive rate for ANA suggests that a genetic factor is involved in SLE pathogenesis. Longitudinal observation of these patients may provide important clinical information and clues to the pathogenesis of SLE.

Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.

During antigen presentation, CD4 functions to stabilize T cell receptor (TCR)-class II MHC interactions and coordinate Ag-induced T cell activation signals. These activation signals cause CD4 down-regulation, presumably acting to optimize T cell activation. We previously reported that oxidative stress interferes with activation-induced CD4 down-regulation in T cells. In this study, we have further investigated inhibition of CD4 down-regulation by oxidative stress and its role for T cell activation. A construct comprised of the mouse FcgammaRIIB extracellular domain and the transmembrane/cytoplasmic domains of human CD4 (FcgammaR/CD4) was expressed in a human T cell line. Oxidant actually potentiated down-regulation of the FcgammaR/CD4 chimera and induced Lck dissociation from both CD4 and FcgammaR/CD4, which is a crucial intracellular process for activation-induced CD4 down-regulation, suggesting a critical role of CD4 ectodomain in the inhibition of CD4 down-regulation by oxidative stress. Furthermore, insertion of CD4 D3-D4 membrane proximal extracellular region between FcgammaR extracellular domain and CD4 transmembrane/cytoplasmic domains in FcgammaR/CD4 chimera made this molecule behave like native CD4 molecule under oxidative stress condition. These data imply that the inhibitory effect of oxidative stress on CD4 down-regulation is executed via D3-D4 domain of CD4 ectodomain. As to its role for T cell activation, CD4 coaggregation with CD3 under the oxidative conditions enhanced activation signal induced by CD3 aggregation. Our results demonstrate that Ag-induced T cell activation which is normally concomitant with CD4 down-regulation may be disturbed through the aberrant regulation of CD4 expression by oxidative stress.

A case of inclusion body myositis with systemic sclerosis.

Abstract We report a case of systemic sclerosis (SSc) associated with inclusion body myositis (IBM). A 58-year-old man was diagnosed as having SSc at the age of 35 years, and had been suffering from chronic progressive weakness and atrophy of the limb muscles. A diagnosis of IBM was established by muscle biopsy. Although most such patients show a poor response to corticosteroids and immunosupressants, glucocorticoid therapy was effective in the present case.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1beta (IL-1beta) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 x g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1beta, and this may contribute to inflammatory reactions in the CNS.

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.

Apoptotic response of eosinophils in chronic eosinophilic pneumonia.

To clarify the pathogenesis of chronic eosinophilic pneumonia (CEP), the apoptosis of eosinophils from bronchoalveolar lavage (BAL-Eos) was compared with that of eosinophils from peripheral blood (PB-Eos) in six cases of CEP. The survival rate of eosinophils and the percentage of apoptotic cells of both types of eosinophils were examined, and the effects of interleukin 5 (IL-5) were evaluated. The role of Fas expression in apoptosis of these eosinophils was also studied. The survival rate of BAL-Eos on the third day of culture was significantly higher than that of PB-Eos (p < 0.01). This was associated with a lower proportion of apoptotic cells in BAL-Eos than in PB-Eos; the percentages of apoptotic cells in PB-Eos and BAL-Eos after 24 h of incubation were 21.7 +/- 3.4% and 10.6 +/- 1.7% respectively. IL-5 suppressed apoptosis and increased the survival rate of both PB-Eos and BAL-Eos. It was found that the apoptotic character of BAL-Eos differed from that of PB-Eos in at least three ways. Firstly, the positive rate of Fas expression on PB-Eos was increased after 24 h of incubation, whereas that on BAL-Eos did not change. Secondly, the expression of Fas on PB-Eos was suppressed by IL-5 (18.5 +/- 4.2% - 8.3 +/- 3.2%, p < 0.05), whereas IL-5 failed to suppress Fas expression on BAL-Eos (3.3 +/- 1.6% - 3.6 +/- 1.0%). Lastly, binding of antibody to Fas antigen induced apoptosis of PB-Eos, but not of BAL-Eos. These data suggested that Fas seemed to be involved in the apoptosis of PB-Eos, whereas BAL-Eos were Fas-resistant in chronic eosinophilic pneumonia. In conclusion, apoptosis of eosinophils might be suppressed by proinflammatory cytokines such as IL-5 leading to their accumulation in the lung. Chronic stimulation of eosinophils in the alveolar space with IL-5 may play a crucial role chronic eosinophilic disorders.

Cell biology of vascular endothelial cells.

The number of molecules identified as being involved in the development of the vascular system has been increasing recently. Among those are secretory molecules and their receptors that collaborate in concert to regulate the process. Of note, however, is that we know little about the response of normal endothelial cells to different stimuli. In this study, we established a method of producing a normal endothelial population from embryonic stem cells. This new culture system was used to analyze the behavior of endothelial cells to various angiogenic stimuli. Our study demonstrated clearly that this culture system is extremely useful in revealing the behavior of developing endothelial cells. Implications derived from our observations are discussed.

Highly specific inhibitory effect of three-component hybrid liposomes including sugar surfactants on the growth of glioma cells.

Three-component hybrid liposomes composed of L-alpha-dimyristoylphosphatidylcholine, micellar surfactant (Tween 20), and beta-D-fructofuranosyl-alpha-D-glucopyranoside monododecanoate were found to be highly effective for inhibiting the growth of glioma cells without any drug.

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.

Human galectin-3 is a novel chemoattractant for monocytes and macrophages.

Galectin-3 is a beta-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 microM) but chemokinetic at low concentrations (10-100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and stromal cell-derived factor-1alpha. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.