Macrophage Chemotactic Activity Research Articles

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75mg/h) monotherapy or co-solvent only (50% hydroxypropyl-β-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Analysis of Macrophage Chemotactic Activity and TLR9 Signaling Pathway in the Mouse Model of Viral Acute Lung Injury.

This study focused on the chemotactic activity of macrophages and the role of the TLR9 signaling pathway in the pathogenesis of viral Acute Lung Injury (ALI). For this purpose, a total of 40 male SPF mice were used, aged 5-8 weeks. They were randomly divided into an experimental group and a control group. The experimental group was further divided into S1 and S2, and the control group was further divided into D1 and D2, with 10 in each. The different groups were detected for the expression of inflammatory cytokines and chemokines and the expression of alveolar macrophages. Results showed that as for the weight, survival status, arterial blood gas analysis, lung index and wet-to-dry value of lung tissue, and lung histopathological analysis results, the S2 group showed more obvious changes versus the D2 group, and the difference was statistically significant (P<0.05). S2 had higher levels of the inflammatory factors TNF-α, IL-1β, IL-6 and the chemokine CCL3 in the BALF supernatant versus the D2 Group, and the difference is statistically significant (P<0.05). S2 had higher expression levels of chemokines CCR5, TLR9, and JMJD1A mRNA versus the D2 group, and the difference was statistically significant (P<0.05). In conclusion, the establishment of a mouse ALI model induced by poly l:C was successful; AM has a certain chemotactic activity on CCL3; polyI:C can promote the expression activity and chemotactic activity of macrophages CCR5 through signal pathways, such as TLR9.

MIP From Legionella pneumophila Influences the Phagocytosis and Chemotaxis of RAW264.7 Macrophages by Regulating the lncRNA GAS5/miR-21/SOCS6 Axis.

BackgroundThe intracellular pathogen Legionella pneumophila (L. pneumophila) is a causative agent of pneumonia and does great harm to human health. These bacteria are phagocytosed by alveolar macrophages and survive to replicate within the macrophages. Despite macrophage infectivity potentiator (MIP) protein serving as an essential virulence factor during the invasion process of L. pneumophila, the regulatory mechanism of MIP protein in the process of bacterial infection to host cells is not yet completely understood. This research thus aims to explore the interaction between MIP and macrophage phagocytosis.MethodsThrough the experiment of the co-culture of RAW264.7 macrophages with different concentrations of MIP, the chemotactic activity of macrophages was detected and the phagocytosis was determined by a neutral red uptake assay. The expression of long noncoding RNA (lncRNA) GAS5, microRNA-21 (miR-21), and suppressor of cytokine signaling (SOCS)6 was determined by qRT-PCR. Target genes were detected by dual luciferase assay.ResultsMIP could reduce the phagocytosis and improve the chemotaxis of RAW264.7 macrophages. The expression of both lncRNA GAS5 and SOCS6 was increased whereas the expression of miR-21 was decreased when macrophages were treated with MIP. Dual luciferase assay revealed that lncRNA GAS5 could interact with miR-21, and SOCS6 served as the target of miR-21. After GAS5 overexpression, the phagocytosis of RAW264.7 treated with MIP was increased whereas the chemotaxis was decreased. In contrast, the opposite results were found in RAW264.7 following GAS5 interference.ConclusionsThe present results revealed that MIP could influence RAW264.7 macrophages on phagocytic and chemotactic activities through the axis of lncRNA GAS5/miR-21/SOCS6.

The role of the thioredoxin/thioredoxin reductase system in the metabolic syndrome: towards a possible prognostic marker?

Mammalian thioredoxin reductase (TrxR) is a selenoprotein with three existing isoenzymes (TrxR1, TrxR2, and TrxR3), which is found primarily intracellularly but also in extracellular fluids. The main substrate thioredoxin (Trx) is similarly found (as Trx1 and Trx2) in various intracellular compartments, in blood plasma, and is the cell's major disulfide reductase. Thioredoxin reductase is necessary as a NADPH-dependent reducing agent in biochemical reactions involving Trx. Genetic and environmental factors like selenium status influence the activity of TrxR. Research shows that the Trx/TrxR system plays a significant role in the physiology of the adipose tissue, in carbohydrate metabolism, insulin production and sensitivity, blood pressure regulation, inflammation, chemotactic activity of macrophages, and atherogenesis. Based on recent research, it has been reported that the modulation of the Trx/TrxR system may be considered as a new target in the management of the metabolic syndrome, insulin resistance, and type 2 diabetes, as well as in the treatment of hypertension and atherosclerosis. In this review evidence about a possible role of this system as a marker of the metabolic syndrome is reported.

Abstract 966: Phase II clinical trial patient responses to the macrophage activating agent RRx-001 correlate to TGF- β pathway activation and markers for fibrosis

Abstract Introduction: RRx-001 is a novel systemically non-toxic small molecule macrophage stimulating agent with promising activity in an ongoing clinical trial in small cell and non-small cell lung cancer and in neuroendocrine tumors. In preclinical studies, RRx-001 activated normally immune-suppressive M2-like Tumor Associated Macrophages (TAMs) to express a series of pro-inflammatory cytokines and chemokines such as transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α TNF-α), inducible nitric oxide synthase (iNOS), and interleukins-10 and -12, a profile that resembles the M1 activated macrophage state. Due to its chemotactic activity for macrophages, TGF-β1 was hypothesized to be involved in the antitumor mechanism of RRx-001. We therefore compared expression patterns of TGF-β1 and TGF-βRI in patient-derived biopsy samples obtained at screening and post RRx-001. As TGF-β1 is closely associated with the induction of fibrosis, we also examined key fibrosis markers. Methods: Tumor biopsies before and after treatment with RRx-001 were obtained from consented patients with NSCLC, SCLC, and neuroendocrine tumors in the QUADRUPLE THREAT Phase II clinical trial (NCT02489903). Post treatment biopsies were obtained following 6 weeks of once-weekly RRx-001 treatment co-incidental with the first on-study CT scan. Tumor samples were evaluated immune-histochemically with putative markers for TGFβ pathway activation (TGF-β1, TGF-βR1), fibrosis (alpha-smooth muscle actin [α-SMA], Matrix metallopeptidase-9 [MMP-9], and Collagen III deposition), and macrophage activation. Patients were subsequently followed for tumor progression. Results: Positive immune-histochemical staining for TGF-β1 and TGF-βR1 was seen in all responding patients, and in none of the non-responding patients to date. In responders we also found that MMP-9 and α-SMA activity was down regulated post RRx-001. The down-regulation of these fibrosis markers is suggestive of less invasive malignancy. The number of tumor associated macrophages and their activation was related to the activity of RRx-001. Conclusions: Our pre and post dose patient derived data to date indicate a correlation between TGF-β signaling activation and a response to RRx-001 that may also correlate with increased numbers of macrophages, and their activation status, in the vicinity of the tumor. These data suggest that activation of the TGF-β1 pathway, as evident by expression of TGF-β1 and TGF-βR1 by tumor cells, could be a predictive biomarker for RRx-001 treatment. Citation Format: Saheli Jha, Thomas A. Summers, Karen Zeman, Christine Brzezniak, Corey Carter, Lindsey Ferry, Jan Scicinski, Bryan Oronsky, Scot Caroen, Jane B. Trepel, Pedro Cabrales, Regina Day. Phase II clinical trial patient responses to the macrophage activating agent RRx-001 correlate to TGF- β pathway activation and markers for fibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 966. doi:10.1158/1538-7445.AM2017-966

A Macrophage NBR1-MEKK3 Complex Triggers JNK-Mediated Adipose Tissue Inflammation in Obesity

The c-Jun NH(2)-terminal kinase (JNK) is a critical determinant of obesity-associated inflammation and glucose intolerance. The upstream mechanisms controlling this pathway are still unknown. Here we report that the levels of the PB1 domain-containing adaptor NBR1 correlated with the expression of proinflammatory molecules in adipose tissue from human patients with metabolic syndrome, suggesting that NBR1 plays a key role in adipose-tissue inflammation. We also show that NBR1 inactivation in the myeloid compartment impairs the function, M1 polarization, and chemotactic activity of macrophages; prevents inflammation of adipose tissue; and improves glucose tolerance in obese mice. Furthermore, we demonstrate that an interaction between the PB1 domains of NBR1 and the mitogen-activated kinase kinase 3 (MEKK3) enables the formation of a signaling complex required for the activation of JNK. Together, these discoveries identify an NBR1-MEKK3 complex as a key regulator of JNK signaling and adipose tissue inflammation in obesity.

Relationship between macrophage differentiation and the chemotactic activity toward damaged myoblast cells

We investigated the effect of macrophage differentiation on the chemotactic activity to invade local damaged myoblasts using in vitro models of muscle injury. We found that: 1) the chemotactic activity of macrophages toward areas of damaged myoblasts might be induced more by live myoblasts than dead ones, 2) the chemotactic activity of macrophages is not due to velocity, but depends on the directionality toward damaged myoblast cells, and 3) macrophage differentiation strongly influence the chemotactic activity toward damaged myoblast cells through the expression of CCR2 and/or F-actin.

Functional characteristics of macrophages in the vitiligo foci

Objective. Study of the chemotactic activity of macrophages and local production of cytokines in sterile skin inflammatory exudate in patients with generalized vitiligo (vitiligo vulgaris). Materials and methods. The authors conducted a study of 22 patients with generalized vitiligo and 22 healthy volunteers. The functional activity of macrophages was assessed with the use of the skin window method based on the method of D.N. Mayansky as amended by V.V. Klimov. The composition of sterile cell exudate was examined with the use of impression smears taken after 6 hours from an epidermis scarification section on the healthy and depigmented skin colored according to the Romanovsky-Gimza method with the use of light microscopy. In addition, cytokines were determined in the supernatant fluid of the skin window exudate from the vitiligo focus obtained by means of centrifugation. Results. The prevalence of mononuclear phagocytes over polymorphonuclear leukocytes was revealed in the depigmentation site, which confirms that macrophages take an active part in the disease pathogenesis (along with a high level of interleukin-18). A low level of IL-10 in the skin window exudate confirms that the suppressor effect in the melanocyte damage zone is weak. These results confirm the important role played by such cells of the immune system as phagocytes in vitiligo pathogenesis, which makes it possible to consider them as potential target cells for developing pathogenetically substantiated approaches to the treatment of the disease.

Stromal MCP‐1 in mammary tumors induces tumor‐associated macrophage infiltration and contributes to tumor progression

There is growing evidence that tumor-associated macrophages (TAMs) promote tumor growth and dissemination. Many individual reports have focused on the protumor function of molecules linked to the recruitment of macrophages, but little is known about which factor has the strongest impact on recruitment of macrophages in breast cancer. To elucidate this question, we performed RT-PCR using species-specific primers and evaluated tumoral and stromal mRNA expression of macrophage chemoattractants separately in human breast tumor xenografts. The correlation between the tumoral or stromal chemoattractant mRNA expression including monocyte chemoattractant protein-1 (MCP-1) (CCL2), MIP-1alpha (CCL3), RANTES (CCL5), colony-stimulating factor 1, tumor necrosis factor alpha, platelet-derived growth factor (PDGF)-BB and macrophage infiltration were compared. There was significant positive correlation between stromal MCP-1 expression and macrophage number (r = 0.63), and negative correlation between tumoral RANTES expression and macrophage number (r = -0.75). However, no significant correlation was found for the other tumoral and stromal factors. The interaction between the tumor cells and macrophages was also investigated. Tumor cell-macrophage interactions augmented macrophage-derived MCP-1 mRNA expression and macrophage chemotactic activity in vitro. Treatment of immunodeficient mice bearing human breast cancer cells with a neutralizing antibody to MCP-1 resulted in significant decrease of macrophage infiltration, angiogenetic activity and tumor growth. Furthermore, immunohistochemical analysis of human breast cancer tissue showed stromal MCP-1 had a significant correlation with relapse free survival (p = 0.029), but tumoral MCP-1 did not (p = 0.105). These findings indicate that stromal MCP-1 produced as a result of tumor-stromal interactions may be important for the progression of human breast cancer and macrophages may play an important role in this tumor-stroma interaction.

Epithelial cells in fetal intestine produce chemerin to recruit macrophages

Macrophages are first seen in the fetal intestine at 11-12 wk and rapidly increase in number during the 12- to 22-wk period of gestation. The development of macrophage populations in the fetal intestine precedes the appearance of lymphocytes and neutrophils and does not require the presence of dietary or microbial antigens. In this study, we investigated the role of chemerin, a recently discovered, relatively selective chemoattractant for macrophages, in the recruitment of macrophage precursors to the fetal intestine. Chemerin mRNA/protein expression was measured in jejunoileal tissue from 10- to 24-wk human fetuses, neonates operated for intestinal obstruction, and adults undergoing bariatric surgery. The expression of chemerin in intestinal epithelial cells (IECs) was confirmed by using cultured primary IECs and IEC-like cell lines in vitro. The regulatory mechanisms involved in chemerin expression were investigated by in silico and immunolocalization techniques. IECs in the fetal, but not mature, intestine express chemerin. Chemerin expression peaked in the fetal intestine at 20-24 wk and then decreased to original low levels by full term. During the 10- to 24-wk period, chemerin accounted for most of the macrophage chemotactic activity of cultured fetal IECs. The maturational changes in chemerin expression correlated with the expression of retinoic acid receptor-beta in the intestine. Chemerin is an important mediator of epithelial-macrophage cross talk in the fetal/premature, but not in the mature, intestine. Understanding the regulation of the gut macrophage pool is an important step in development of novel strategies to boost mucosal immunity in premature infants and other patient populations at risk of microbial translocation.

The macrophage chemotactic activity of Edwardsiella tarda extracellular products

The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection.

Fas‐mediated upregulation of vascular endothelial growth factor and monocyte chemoattractant protein‐1 expression in cultured dermal fibroblasts: Role in the inflammatory response

The Fas-Fas ligand interaction is the most important pathway in starting apoptosis. In addition, several recent reports have emerged documenting non-apoptotic roles for Fas. However, a non-apoptotic role of Fas in dermal fibroblasts remains unknown. The present study investigated whether Fas stimulation not only promotes apoptosis but also stimulates elements of the inflammatory response such as angiogenesis and macrophage infiltration. Fas stimulation was performed by treating cultured human dermal fibroblasts with an agonistic anti-Fas monoclonal antibody (mAb). Anti-Fas mAb-treated fibroblasts showed a significantly greater increase of caspase-3 and caspase-8 activity compared with control fibroblasts. Addition of the anti-Fas mAb induced DNA fragmentation, as confirmed by the DNA ladder assay. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining showed that treatment with the anti-Fas mAb induced an increase of apoptotic fibroblasts in a time-dependent manner. At both mRNA and protein levels, anti-Fas mAb-treated fibroblasts showed significantly higher expression of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP)-1 compared with control fibroblasts. A pan-caspase inhibitor (Z-VAD-FMK) significantly inhibited VEGF and MCP-1 expression. After transplantation of fibroblasts into mice with severe combined immunodeficiency, the nodules derived from anti-Fas mAb-treated fibroblasts showed more abundant neovascularization, increased macrophage infiltration, and more apoptotic cells in comparison with nodules derived from control fibroblasts. The results of both in vitro and in vivo studies confirmed significantly higher angiogenic activity and macrophage chemotactic activity of anti-Fas mAb-treated fibroblasts compared with control fibroblasts.

Immunosuppressive Factors Secreted by Human Amniotic Epithelial Cells

Amniotic membrane has been applied to the ocular surface to restore corneal function. The beneficial effect of amniotic membrane transplantation may be due to the immunosuppressive effects of amniotic epithelial cells. The purpose of this study was to determine whether amniotic epithelial cells (AECs) secrete anti-inflammatory and antiproliferative factors that affect the chemotaxis of neutrophils and macrophages and suppress both T- and B-cell proliferation in vitro. Human amniotic cells were isolated from human amniotic membrane and cultured in vitro. The supernatants from AEC cultures were collected after 48 hours of incubation. Neutrophil and macrophage chemotactic activity was tested in the presence of AEC supernatant, using 24-well migration assay chambers. Lymphocyte proliferation was tested by H(3)-thymidine incorporation. Apoptosis was examined by caspase-3 and annexin V assays, and expression of cytokines was assessed by RT-PCR. AEC supernatant significantly inhibited the chemotactic activity of neutrophils and macrophages toward macrophage inflammatory protein (MIP)-2 (P < 0.05). The supernatant significantly reduced the proliferation of both T and B cells after mitogenic stimulation (P < 0.05). Caspase-3 assays revealed that the supernatant induced apoptosis of T and B cells, but not of corneal epithelial cells and liver cells. In contrast to lymphocytes, macrophages and neutrophils were resistant to apoptosis induced by AEC supernatant. The AECs expressed message for TNFalpha, Fas ligand (FasL), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), TGFbeta, and macrophage migration-inhibitory factor (MIF). However, AEC induction of apoptosis was inhibited (50%) by anti-FasL antibody but not by anti-TRAIL or anti-TNFalpha antibodies. Moreover, AEC supernatant inhibited macrophage migration in vitro. AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems.

Propionibacterium acnes-induced hepatic granuloma formation is impaired in mice lacking tetraspanin CD9

The granuloma is a host defence response to persistent pathogenic irritants. In the process of granuloma formation, the activation, migration, and fusion of macrophages occur locally, but the mechanisms involved remain elusive. Tetraspanins regulate cell migration and fusion by organizing functional molecular complexes in membrane microdomains. Here we investigated the role of tetraspanin CD9 in hepatic granuloma formation. Immunostaining of the liver of untreated wild-type mice showed that CD9 was expressed by vascular endothelial cells and perivenular hepatocytes. When intrahepatic granulomas were induced by intravenous injection of Propionibacterium acnes, hepatocyte CD9 was extensively upregulated, while inflammatory cells constituting granulomas were mostly negative for CD9. Compared with wild-type littermates, CD9-knockout mice showed dissemination of Propionibacterium acnes and reduced number and size of granulomas after the injection. Moreover, production of granuloma-inducing cytokines, TNF-alpha and IFN-gamma, was delayed and chemotactic activity for macrophages was suppressed in the liver of mutant mice. These results suggest that CD9 is one of the proteins that promotes granuloma formation in the liver.

Periprosthetic osteolysis: induction of vascular endothelial growth factor from human monocyte/macrophages by orthopaedic biomaterial particles.

VEGF and VEGF receptor, Flt-1, expression was observed in periprosthetic tissues surrounding loosened total joint implants. Exposure of monocyte/macrophages to titanium particles resulted in increased VEGF expression, p44/42 MAPK activation, and VEGF-dependent macrophage chemotaxis. Increased levels of angiogenic factors, such as VEGF, may be critically important in wear debris-induced implant loosening after total joint arthroplasty. Periprosthetic osteolysis after total hip arthroplasty occurs in association with formation of a vascularized granulomatous tissue in response to particulate debris. This study examined expression of vascular endothelial growth factor (VEGF) and the VEGF receptor in 10 periprosthetic tissues from loosened prostheses and quantified effects of titanium particles on VEGF release, intracellular signaling, and VEGF-dependent chemotaxis in primary cultures of human monocyte/macrophages. Double immunofluorescent staining showed that VEGF and Flt-1 co-localized with cells positive for the macrophage marker, CD11b, in the periprosthetic tissues. Monocyte/macrophages challenged with titanium particles showed a dose- and time-dependent release of VEGF ranging from 2.8- to 3.1-fold and exhibited increased expression of VEGF121 and VEGF165 mRNAs, reaching levels up to 5.0- and 8.6-fold, respectively, by 48 h (p < 0.01). Exposure of monocyte/macrophages to titanium particles upregulated phosphorylated-p44/42 mitogen-activated protein kinase (MAPK) within 30 minutes. Particle-induced activation of p44/42 MAPK and release of VEGF were dose-dependently suppressed by pretreatment of cells with PD98059, a specific inhibitor of p44/42 MAPK. Monocyte/macrophages challenged with titanium particles also showed a time-dependent activation of AP-1, a transcription factor associated with VEGF expression (p < 0.01). Supernatants from particle-challenged monocyte/macrophages increased macrophage chemotactic activity by 30%, which was significantly inhibited by anti-VEGF neutralizing antibody (p < 0.01). This study suggests that induction of VEGF release from monocyte/macrophages in response to orthopaedic biomaterial wear debris may contribute to periprosthetic osteolysis and implant loosening.

Signal transduction pathways involved in macrophage migration induced by peritoneal fluid chemotactic factors in stages I and II endometriosis

Objective: To explore the role of G-protein coupled signaling pathways in activation of macrophage migration in endometriosis stages I and II. Design: Case controlled study. Setting: University hospital. Patient(s): Fifteen patients undergoing laparoscopy for elective sterilization (n = 5) or for diagnosis of endometriosis stages I and II associated with infertility (n = 10). Intervention(s): Peritoneal fluid samples were collected during laparoscopy. Main Outcome Measure(s): Macrophage migration induced by peritoneal fluid from patients with endometriosis stages I and II (PF SI-II) and potential G-protein coupled receptors and second messengers involved in macrophage activation. Result(s): Potential G-protein coupled receptors and second messengers involved in macrophage activation were evaluated after incubation of U-937 cells differentiated into macrophages with inhibitors of phospholipase A and C, adenylate cyclase, and protein kinase A and C. Macrophage chemotactic activity induced by PF SI-II was inhibited in the presence of a phospholipase C and A 2 inhibitor (IC 50= 30 μM) and after treatment with myristoylated protein kinase C peptide inhibitor (50 nM). An increase in inositol phosphate (IP 3) was also observed in macrophages exposed to PF SI-II. Activation of multiple G-proteins in macrophages was examined after exposure of cells to PF SI-II in the presence and absence of Bordettela pertussis and cholera toxins. No effect on macrophage migration was observed. Conclusion(s): Macrophage chemotaxis induced by PF SI-II appears to involve activation of pertussis toxin-insensitive G-protein coupled receptors in macrophages. Our data suggest that these events lead to subsequent activation of phospholipases followed by generation of IP 3 and potential mobilization of intracellular Ca 2+. Subsequent phosphorylation of target proteins by protein kinase C may regulate the chemotactic responses. The adenylate cyclase pathway does not appear to play a role in this process.

A novel immunostimulating aspect of Lactobacillusgasseri: induction of “Gasserokine” as chemoattractants for macrophages

The chemotactic activity of the culture supernatants from 14 strains of Lactobacillus acidophilus and L. gasseri was examined for murine macrophages. Significant macrophage chemotactic activity was observed in three strains of L. acidophilus and all strains of L. gasseri. The highest activity was observed in the supernatant (1131-sup) from 24-h cultures of L. gasseri JCM1131 T. The chemotactic factor from 1131-sup, designated as “Gasserokine”, was purified by the C 18 reverse phase and ion-exchange chromatography. The purity of Gasserokine was checked by HPLC with the reverse-phase mode. The chemotactic activity of Gasserokine was also observed for human monocytes. The macrophage chemotaxis induced by L. gasseri JCM1131 T culture supernatants was discovered to be a new biological function exerted by probiotic lactic acid bacteria. Therefore, the activity is expected to be used for one of the functional parameters in the immunomodulating properties of probiotic lactic acid bacteria.

In vitro Leishmania major promastigote-induced macrophage migration is modulated by sensory and autonomic neuropeptides.

Recruitment, migration and adherence of macrophages and their interaction with inoculated promastigotes are key steps in the initiation of the inflammatory process in cutaneous leishmaniasis. Parasite- and nervous system-derived factors might be involved in this process. In the present study the chemotactic activities of live, killed and sonicated Leishmania major promastigotes and of the promastigote culture supernatant as well as the L. major surface protease gp63 towards a murine macrophage cell line, Raw 264.7, were investigated, using the Boyden technique. The sensory neuropeptides SOM, CGRP and SP, and the autonomic neuropeptides VIP and NPY, were also investigated for possible modulatory effects on this chemotaxis, using the living promastigotes. Living promastigotes were the most efficient attractants for macrophages compared with other forms of the parasites. Prior incubation of the macrophages with the parasites completely abolished the chemotactic activity. This might indicate that the living promastigote chemotaxis is a receptor-mediated process. On the other hand, paraformaldehyde-killed promastigotes not only failed to induce macrophage chemotaxis but also inhibited it in comparison with the control. The surface protease gp63 tended to inhibit the macrophage chemotactic activity and the sonicate tended to stimulate it compared with controls. The culture supernatant had no effect, indicating that the chemoattractive factors putatively synthesized by the living promastigotes are not released to the surrounding medium. Somatostatin inhibited L. major promastigote-induced macrophage migration at a high concentration, 10(-6) M, while substance P inhibited it at both low concentrations, 10(-10) and 10(-9) M, and a high one, 10(-6) M, the last-mentioned having the greatest inhibitory effect. A stimulatory effect of calcitonin gene-related peptide was found at high concentrations, 10(-5) and 10(-6) M. Vasoactive intestinal peptide stimulated macrophage chemotactic activity at both a high, 10(-5) M, and at a low, 10(-9) M, concentration, the same concentration at which neuropeptide Y exerted its maximum inhibitory effect.

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice.

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells.

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.