Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: a quid pro quo in pancreatic cancer.

INTRODUCTION: More effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model. METHODS: 6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays. RESULTS: Clodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm(3) to 92.3 mm(3) (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034). CONCLUSIONS: This quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.

Macrophages promote tumour growth and liver metastasis in an orthotopic syngeneic mouse model of colon cancer.

PURPOSE: Tumour-associated macrophages have been shown to promote proliferation, angiogenesis and metastasis in several carcinomas. The effect on colon cancer has not yet been clarified. Furthermore, Kupffer cells in the liver might initiate the formation of metastases by directly binding tumour cells. METHODS: An orthotopic syngeneic mouse model of colon cancer as well as a liver metastases model has been studied, using murine CT-26 colon cancer cells in Balb/c-mice. Macrophages were depleted in both models by clodronate liposomes. Tumour sizes and metastases were determined using 7-Tesla MRI. The macrophage and vascular density in the orthotopic tumours as well as the Kupffer cell density in the livers were evaluated using immunohistochemistry. RESULTS: Animals in the macrophage-depleted group displayed significantly smaller primary tumours (37 ± 20 mm(3)) compared to the control group (683 ± 389 mm(3), p = 0.0072). None of the mice in the depleted group showed liver or peritoneal metastases, whereas four of six control mice displayed liver and five out of six mice peritoneal metastases. The vascular density was significantly lower in the macrophage-depleted group (p = 0.0043). In the liver metastases model, animals of the Kupffer cell-depleted group (14.3 ± 7.7) showed significantly less liver metastases than mice of the two control groups (PBS liposomes, 118.5 ± 28.2, p = 0.0117; NaCl, 81.7 ± 23.2, p = 0.0266). The number of liver metastases correlated directly with the Kupffer cell density (p = 0.0221). CONCLUSION: Macrophages promote tumour growth, angiogenesis and metastases in this orthotopic syngeneic mouse model. Kupffer cells enhance the formation of metastases in the liver.

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice.

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.

Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion.

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.

Minocycline reduces glioma expansion and invasion by attenuating microglial MT1-MMP expression.

Glioma cells release soluble factors, which induce the expression of membrane type 1 matrix metalloprotease (MT1-MMP) in tumor associated microglia and then exploit MT1-MMP mediated matrix degradation for invasion. Here, we show that minocycline blocked the increase in MT1-MMP expression and activity in cultivated microglia stimulated with glioma conditioned medium. Glioma growth within an organotypic brain slice preparation was reduced by minocycline and this reduction depended on the presence of microglia. Glioma growth in an experimental mouse model was strongly reduced by the addition of minocycline to drinking water, compared to untreated controls. Coherently, we observed in our orthotopic glioma implantation model, that MT1-MMP was abundantly expressed in glioma associated microglia in controls, but was strongly attenuated in tumors of minocycline treated animals. Overall, our study indicates that the clinically approved antibiotic minocycline is a promising new candidate for adjuvant therapy against malignant gliomas.

Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice.

A single intracheal dose of liposome-encapsuled dichloro-methylene-diphosphonate resulted in the elimination of alveolar macrophages (AM) from the lung, creating a model to study the in vivo role of AM in the pulmonary immune response. Using intratracheally administered trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), the kinetics of the response, the location and number of TNP-specific antibody-forming cells, and the different Ig classes of the antibodies produced were studied in AM-depleted animals. The results show that AM elimination has a dramatic effect on the pulmonary immune responses against TNP-KLH. An increase in APC in lung-associated lymph nodes and a prolongation of the response is found, as well as an introduction of APC in lung tissue. In both experimental groups, the majority of the TNP-specific antibodies produced was IgG, followed by IgA and IgE, while very few IgM antibodies could be detected. We conclude from these results that AM are likely to play a role in controlling the pulmonary immune response in a suppressive way, thereby limiting the possible damage caused by severe immune responses in lung tissue.

Distribution and kinetics of superantigen-induced cytokine gene expression in mouse spleen.

The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain staphylococcal and streptococcal infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal BALB/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for interleukin 2 (IL-2), interferon gamma, and tumor necrosis factors (TNF) alpha and beta were not expressed at detectable levels in spleens of unstimulated animals but became visible already 30 min after intraperitoneal application of 50 micrograms staphylococcal enterotoxin B. All mRNA levels showed peak expression approximately 3 h after injection and a slow decrease up to 24 h after injection. Expression of the mRNAs was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets of the spleen. Interestingly, TNF-alpha mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-alpha mRNA showed a broader distribution indicating a second cell population producing TNF-alpha. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage-depleted mice. Only the second phase of TNF-alpha mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of cyclosporin A. These data show that nonphagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF-alpha is T cell derived.

Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages.

Almost 50% of the cells infiltrating the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) are macrophages (M psi). To investigate the role of the M psi in the pathogenesis of EAE, we eliminated M psi by means of mannosylated liposomes containing dichloromethylene diphosphonate (Cl2MDP). Cl2MDP-containing liposomes injected intravenously eliminate M psi in spleen and liver. Incorporation of mannose into the lipid layers enables the liposomes to pass the blood-brain barrier (BBB). Injections of Cl2MDP-containing mannose liposomes intravenously shortly before the appearance of clinical signs, markedly suppressed the expression of clinical signs of EAE. This suppression was accompanied by a marked reduction of infiltrated M psi in the CNS. Cl2MDP-containing liposomes without mannose incorporated had no effect. Cl2MDP-containing mannosylated liposomes had no effect on plasma corticosterone levels compared with injections of saline; thus, the suppression of expression of EAE was not corticosterone mediated. These results show that the M psi within the CNS play an important role in the pathogenesis of EAE.

Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum.

We have recently described that sustained Plasmodium falciparum growth could be obtained in immunodeficient mice. We now report the potential of this new mouse model by assaying the effect of the passive transfer of antibodies (Abs) which in humans have had a well-established effect.Our results show that the total African adult hyperimmune immunoglobulin Gs (HI-IgGs) strongly reduce P. falciparum parasitemia similarly to that reported in humans, but only when mice are concomitantly reconstituted with human monocytes (HuMNs). In contrast, neither HI-IgGs nor HuMNs alone had any direct effect upon parasitemia. We assessed the in vivo effect of epitope-specific human Abs affinity-purified on peptides derived either from the ring erythrocyte surface antigen (RESA) or the merozoite surface protein 3 (MSP3). The inoculation of low concentrations of anti-synthetic peptide from MSP3, but not of anti-RESA Abs, consistently suppressed P. falciparum in the presence of HuMNs. Parasitemia decrease was stronger and faster than that observed using HI-IgGs and as fast as that induced by chloroquine. Our observations demonstrate that this mouse model is of great value to evaluate the protective effect of different Abs with distinct specificity in the same animal, a step hardly accessible and therefore never performed before in humans.

The role of macrophages in T cell-mediated autoimmune diabetes in nonobese diabetic mice.

We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into beta cell-cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their beta cell-cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill beta cells, we examined the islet antigen-specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells that cause beta cell destruction, resulting in autoimmune diabetes in NOD mice.

Role of brain macrophages on IL-1beta and fatigue following eccentric exercise-induced muscle damage.

Fatigue associated with recovery from muscle damage has recently been linked to increases in brain and muscle proinflammatory cytokines. However, little is known regarding the origin of these cytokines. Since macrophage-like cells in the brain are a primary source of cytokines, we used a brain specific macrophage depletion technique involving liposome encapsulated clodronate (CLD) to examine the role of macrophages on brain IL-1beta and fatigue following eccentric exercise-induced muscle damage. Mice were assigned to six groups: Downhill saline (DWNSAL), downhill clodronate (DWNCLD), uphill saline (UPSAL), uphill clodronate (UPCLD), non-running saline (CONSAL) or non-running clodronate (CONCLD). Mice were given intracerebroventricular (ICV) (10 microL) injections of clodronate-filled liposomes (CLD) to deplete macrophages, or saline-filled liposomes (SAL) and run on a treadmill at 22m/min and -14% (DWN) or 14% (UP) grade for 150 min. A subset of uphill and downhill running mice (n=40) was then run to fatigue on a treadmill at 36m/min, 8% grade at 24h after the uphill and downhill runs. A second subset of uphill, downhill, and control mice (n=30) was sacrificed 24h after the run for analysis of brain IL-1beta concentration. Histological examination confirmed previous reports that CLD administration reduced perivascular and meningeal macrophage subsets in the brain. CLD reduced IL-1beta concentration in the cortex of DWN mice (P<0.05), which was associated with enhanced treadmill performance 24h after both uphill and downhill runs (P<0.05) although the magnitude was greater following the downhill run. These results suggest that brain macrophages can contribute to the increase in brain IL-1beta and fatigue that are associated with recovery from exercise-induced muscle damage.

Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40.

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

Increased frequency of CD16+ monocytes and the presence of activated dendritic cells in salivary glands in primary Sjögren syndrome.

OBJECTIVES: In the salivary glands of patients with primary Sjögren Syndrome (pSjS) an accumulation of dendritic cells (DCs) is seen, which is thought to play a role in stimulating local inflammation. Aberrancies in subsets of monocytes, generally considered the blood precursors for DCs, may play a role in this accumulation of DCs. This study is aimed at determining the level of mature CD14lowCD16+ monocytes in pSjS and their contribution to the accumulation of DCs in pSjS. METHODS: Levels of mature and immature monocytes in patients with pSjS (n = 19) and controls (n = 15) were analysed by flow cytometry. The reverse transmigration system was used for generation of DCs generated from monocyte subsets. The phenotype of DCs in pSjS salivary glands was analysed using immunohistochemistry. In vivo tracking of monocyte subsets was performed in a mouse model. RESULTS: Increased levels of mature CD14lowCD16+ monocytes were found in patients with pSjS (mean (SD) 14.5 (5.5)% vs 11.4 (3.4)%). These cells showed normal expression of chemokine receptor and adhesion molecules. Mature monocytes partly developed into DC-lysosome-associated membrane glycoprotein (LAMP)+ (19.6 (7.5)%) and CD83+ (16 (9)%) DCs, markers also expressed by DCs in pSjS salivary glands. Monocyte tracking in the non-obese diabetic (NOD) mouse showed that the homologue population of mature mouse monocytes migrated to the salivary glands, and preferentially developed into CD11c+ DCs in vivo. CONCLUSIONS: Mature monocytes are increased in pSjS and patient and mouse data support a model where this mature monocyte subset migrates to the salivary glands and develops into DCs.

Neutrophil degranulation mediates severe lung damage triggered by streptococcal M1 protein.

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome commonly associated with acute lung injury. The aim of the present study was to investigate the role of neutrophils and their secretion products in M1 protein-induced lung damage. The degranulation of neutrophils by M1 protein was studied in whole blood using marker analysis for individual granule subsets. In mice, M1 protein was injected intravenously and the lung damage was assessed by histology, electron microscopy, cell count in bronchoalveolar lavage fluid and analysis of lung vascular permeability. Comparisons were made in mice with intact white blood count, neutropenic mice and neutropenic mice injected with the secretion of activated neutrophils. In whole blood, M1 protein forms complexes with fibrinogen that bind to beta(2)-integrins on the neutrophil surface, resulting in degranulation of all four subsets of neutrophil granules. Intravenous injection of M1 protein into mice induced neutrophil accumulation in the lung, increase in vascular permeability and acute lung damage. Depletion of neutrophils from the circulation completely abrogated lung injury and vascular leakage. Interestingly, the lung damage was restored by injecting neutrophil secretion. The present data suggest that neutrophil granule proteins are directly responsible for lung damage induced by the streptococcal M1 protein.

Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension.

Intraocular pressure (IOP) elevation has often been used as an experimental model to study mechanisms underlying retinal ganglion cell (RGC) death associated with ocular ischemic injury and glaucoma. The aim of the present study, using both in vitro and in vivo approaches, was to investigate the role of phosphatidylinositol 3-kinase (PI3K)/akt pathway in RGC viability in normal rats and rats following transient IOP elevation. For in vivo studies, pathway inhibitors were administered intravitreally on days 3, 9, and 15 post-2-h IOP elevation at 110 mm Hg. Toward the end of the 3-week examination period, the fluorescent dye Fluorogold was used to retrogradely label surviving RGCs. In order to examine the role of macrophages that were recruited into the eye following the pathway inhibition, clodronate liposomes were used to deplete phagocytic cells in the eye. PI3K/akt pathway activity and location in the retina were examined using Western blot and immunohistochemistry, respectively. Here we showed that PI3K/akt inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and KY12420 at low concentrations (2 microM or 20 microM) did not influence RGC survival but caused RGC loss at high concentration (200 muM) in retinal explants derived from intact rats. In contrast, both LY294002 and KY12420 at 20 microM led to RGC loss in retinal explants derived from IOP-elevated eyes. A detrimental action of phagocytic cells on RGC survival was also seen in these retinas. In vivo results confirmed the detrimental actions of PI3K/akt inhibition and macrophages on RGC survival in IOP-elevated, but not intact eyes even with high concentration of LY294002. Low level of PI3K/akt activity was detected in the ganglion cell layer (GCL) in intact retina. Acute IOP elevation activated PI3K/akt pathway in the inner nuclear layer and GCL including RGCs. This study thus demonstrates that PI3K/akt pathway mediates RGC survival after IOP elevation but not under normal condition.

Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages.

Eosinophils are associated with allergic diseases and helminth infections. Development of these cells and recruitment to peripheral tissues are only partially understood. Distinct stages of eosinophil development in fetal liver, bone marrow, and blood could be identified using IL-4 reporter mice and mAb against FIRE, Siglec-F, and CCR3. Immature eosinophils were present in the fetal liver and could reconstitute the eosinophil compartment in irradiated recipient mice. In adult mice, eosinophil maturation proceeded from CCR3(-) to CCR3(+) cells in the bone marrow and was accompanied with changes in the transcriptional profile. Eosinophils appeared as activated cells in lung, thymus, lymph nodes, and Peyer's patches but remained in a resting state in bone marrow, blood, and spleen. Mixed bone marrow chimeras revealed that recruitment to lung and peritoneum was dependent on Stat6 expression in noneosinophils. Alternatively activated macrophages contributed substantially to tissue recruitment of eosinophils, providing a novel basis for development of therapeutic approaches to lower tissue eosinophilia.

Macrophage-mediated neuroprotection and neurogenesis in the olfactory epithelium.

Resident and recruited olfactory epithelial macrophages participate in the regulation of the survival, degeneration, and replacement of olfactory sensory neurons (OSNs). We have reported that liposome-encapsulated clodronate (Lip-C) induced selective and statistically significant depletion of macrophages in the OE of sham and 48 h OBX mice (38 and 35%, respectively) that resulted in increased OSN apoptosis and decreased numbers of mature OSNs and proliferating basal cells compared to controls (Lip-O). The aim of this study was to identify molecular mechanisms by which the selective depletion of macrophages in the OE resulted in these cellular changes by using a microarray expression pattern analysis. A 2x2 ANOVA identified 4,085 overall significantly (P < 0.01) regulated genes in the OE of Lip-O and Lip-C sham and 48 h OBX mice, and further statistical analysis using pairwise comparisons identified 4,024 genes that had either a significant (P < 0.01) treatment main effect (n = 2,680), group main effect (n = 778), or interaction effect (n = 980). The mean hybridization signals of immune response genes, e.g., Cxcr4, and genes encoding growth factors and neurogenesis regulators, e.g., Hdgf and Neurod1, respectively, were primarily lower in Lip-C mice compared with Lip-O mice. Apoptosis genes, e.g., Bak1, were also differentially regulated in Lip-C and/or OBX mice. Expression patterns of selected genes were validated with real-time RT-PCR; immunohistochemistry was used to localize selected gene products. These results identified the differential regulation of several novel genes through which alternatively activated macrophages regulate OSN progenitor cell proliferation, differentiation, and maturation, and the survival of OSNs.

Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity.

Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K(+) concentration. We report here that incubation of sporozoites to the intracellular K(+) concentration enhances 8-10 times the infectivity of Plasmodium berghei and 4-5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K(+) enhancing effect was time and concentration dependent, and was significantly decreased by K(+) channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K(+) treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K(+) treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

The effect of elimination of macrophages on the tissue distribution of liposomes containing [3H]methotrexate.

In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.