Activation State Of Macrophages Research Articles

Implications of immunomodulatory interleukins for the hyperimmunoglobulinemia of Sjögren’s syndrome

A prospective study of 37 patients with pSS and 20 healthy controls was performed to analyze the differences in circulating levels of macrophage-derived and Th1/Th2 cytokines which could explain the hyperimmunoglobulinemia, characteristic of primary Sjögren’s syndrome (pSS). Serum levels of interleukin (IL)-6, IL-10, IL-12, gamma-interferon (γ-INF) and IL-4 were analyzed by a sandwich immunoassay-based protein array system. When compared with the control group, higher levels of IL-6, IL-12 and IL-10 and a lower Th1/Th2 ratio, as demonstrated by the γ-INF/IL-4 ratio, were detected in patients. The levels of IL-4 were notably higher in pSS patients with monoclonal gammopathy. Serum IL-4 and IL-10 levels and immunoglobulin G concentrations were significantly correlated. In conclusion, patients with pSS show a state of macrophage and T-lymphocyte activation with increased concentrations of cytokines implicated in the differentiation of B cells and secretion of immunoglobulins.

Modulation of Macrophage Activation State Protects Tissue from Necrosis during Critical Limb Ischemia in Thrombospondin-1-Deficient Mice

BackgroundMacrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.Methods and FindingsUsing a genetic model of tsp-1 −/− mice subjected to femoral artery excision, we report that tsp-1 −/− mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1 −/− and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1 −/− mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1 −/− mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1 −/− mice, thereby demonstrating that macrophages mediated tissue protection in these mice.ConclusionThis study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Activation of Alveolar Macrophages after Plutonium Oxide Inhalation in Rats: Involvement in the Early Inflammatory Response

Alveolar macrophages play an important role in the distribution, clearance and inflammatory reactions after particle inhalation, which may influence long-term events such as fibrosis and tumorigenesis. The objectives of the present study were to investigate the early inflammatory events after plutonium oxide inhalation in rats and involvement of alveolar macrophages. Lung changes were studied from 3 days to 3 months after inhalation of PuO2 of different isotopic compositions (70% or 97% 239Pu) and initial lung deposits (range 2.1 to 43.4 kBq/rat). Analyses of bronchoalveolar lavages showed early increases in the numbers of granulocytes, lymphocytes and multinucleated macrophages. The activation of macrophages was evaluated ex vivo by measurement of inflammatory mediator levels in culture supernatants. TNF-alpha and chemokine MCP-1, MIP-2 and CINC-1 production was elevated from 7 days after inhalation and remained so up to 3 months. In contrast, IL-1beta, IL-6 and IL-10 production was unchanged. At 6 weeks, pulmonary macrophage numbers and activation state were increased as observed from an immunohistochemistry study of lung sections with anti-ED1. Similarly, histological analyses of lung sections also showed evidence of inflammatory responses. In conclusion, our results indicate early inflammatory changes in the lungs of PuO2-contaminated animals and the involvement of macrophages in this process. A dose-effect relationship was observed between the amount of radionuclide inhaled or retained at the time of analysis and inflammatory mediator production by alveolar macrophages 14 days after exposure. For similar initial lung deposits, the inflammatory manifestation appears higher for 97% 239Pu than for 70% 239Pu.

Unfolding the relationship between secreted molecular chaperones and macrophage activation states.

Over the last 20 years, it has emerged that many molecular chaperones and protein-folding catalysts are secreted from cells and function, somewhat in the manner of cytokines, as pleiotropic signals for a variety of cells, with much attention being focused on the macrophage. During the last decade, it has become clear that macrophages respond to bacterial, protozoal, parasitic and host signals to generate phenotypically distinct states of activation. These activation states have been termed 'classical' and 'alternative' and represent not a simple bifurcation in response to external signals but a range of cellular phenotypes. From an examination of the literature, the hypothesis is propounded that mammalian molecular chaperones are able to induce a wide variety of alternative macrophage activation states, and this may be a system for relating cellular or tissue stress to appropriate macrophage responses to restore homeostatic equilibrium.

Piroxicam, indomethacin and aspirin action on a murine fibrosarcoma. Effects on tumour-associated and peritoneal macrophages

Growth of a methylcholanthrene-induced fibrosarcoma in BALB/c mice was accompanied by an increase in the activation state of tumour-associated macrophages (TAM), as measured by their FcIgG receptor expression, phagocytic index and beta-glucuronidase levels. All of these parameters were markedly higher in TAM than in peritoneal macrophages (PM) derived from the same animal. On the other hand, PM from tumour-bearing mice showed lower activation parameters than PM from normal animals. We also studied the effect on tumour development of three inhibitors of prostaglandin synthesis: indomethacin, piroxicam and aspirin. Intraperitoneal administration of these drugs during 8 d was followed by the regression of palpable tumours. Indomethacin (90 mg/d) induced 45% regression, while with piroxicam (two 400 mg/d doses and six 200 mg/d doses) and aspirin (1 mg/d) 32% and 30% regressions, respectively, were observed. The growth rate of nonregressing tumours, which had reached different volumes by the end of the treatment, was delayed to a similar extent by the three anti-inflammatory non-steroidal drugs (NSAID). With respect to TAM, the treatment did not induce any significant change in their activation state, though both piroxicam and indomethacin increased slightly the TAM number. In contrast, NSAID administration was followed by a remarkable increase in the activation parameters of PM when compared with PM from tumour-bearing mice receiving no treatment. Indeed, these parameters were in some cases higher than those of PM from normal mice. The leukocytosis (60,000/microliters) with neutrophilia (80%) induced by tumour growth on peripheral blood leukocytes (PBL) was reversed by the treatment to values close to normal, in parallel with the reduction of tumour size. A drop in haematocrit was also noted which was most probably a consequence of tumour growth rather than of the treatment. This study reveals that the three NSAID tested have a remarkable antitumour activity, which correlates with the restoration of PM activity and PBL values.

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.

Low dose streptozotocin causes stimulation of the immune system and of anti-islet cytotoxicity in mice.

Multiple low doses of streptozotocin are known to induce immune-mediated insulin deficient diabetes and depression of immune reactivity. We show here that immune depression by streptozotocin is not general but that some parts of the immune system are stimulated. Spleen cells from streptozotocin-treated mice showed enhanced cytotoxicity against syngeneic islet cells and various tumour cells including insulinoma cells. Several cell types served as effector cells, including macrophages, asialo GM1+ and Lyt-2+ lymphocytes. The increased cytotoxic activity towards islet cells was mostly due to macrophages and to non-asialo GM1+ and non-Lyt-2+ lymphocytes. A higher activation state of macrophages in low dose streptozotocin-treated mice was demonstrated by measurements of superoxide anion release. We conclude that multiple low doses of streptozotocin stimulate 'natural cytotoxicity', i.e. the non-MHC restricted cytotoxic activity of macrophages, T cells and natural killer lymphocytes.

Increased serum neopterin in patients with HIV-1 infection is correlated with reduced in vitro interleukin-2 production

Recently we have observed that the CD4+ T cell response of peripheral blood mononuclear cells (PBMC) to soluble antigens is the first to be lost in the course of HIV-1 infection followed by the loss of response to HLA alloantigens. In this study we compared serum neopterin concentrations of individuals with early stages of HIV-1 infection (stages WR1 and WR2, Walter Reed staging system) with in vitro interleukin-2 (IL-2) production of PBMC in response to stimulation with soluble antigens (influenza A virus and tetanus toxoid) and alloantigens. Neopterin concentrations were significantly higher in HIV-1-seropositive individuals who showed deficient IL-2 production in response to recall antigens only or to all of the stimuli tested in vitro, compared with HIV-1-seropositive individuals who exhibited no CD4+ T cell defects. No difference in serum neopterin concentrations was observed between the group that was functionally deficient to soluble antigens only versus those who were unresponsive to both types of stimuli. It appears that the selective loss of the MHC self-restricted CD4+ T cell function is associated with an increase in serum neopterin levels. Neopterin concentrations are an estimate of the activation status of macrophages. We conclude that defective in vitro production of lymphokines by T lymphocytes is associated with activated macrophages in vivo.

Chronic Estradiol Administration In Vivo Promotes the Proinflammatory Response of Macrophages to TLR4 Activation: Involvement of the Phosphatidylinositol 3-Kinase Pathway

Short-term exposure to 17beta-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor alpha (ERalpha)-dependent activation of the PI3K pathway. In the present study, we confirm that in vitro exposure of mouse peritoneal macrophages to E2 enhanced Akt phosphorylation and slightly decreased LPS-induced cytokine production. In striking contrast, we show that chronic administration of E2 to ovariectomized mice markedly increases the expression of IL-1beta, IL-6, IL-12p40, and inducible NO synthase by resident peritoneal macrophages in response to LPS ex vivo. These results clearly indicate that short-term E2 treatment in vitro does not predict the long-term effect of estrogens in vivo on peritoneal macrophage functions. We show that this in vivo proinflammatory effect of E2 was mediated through ERalpha. Although the expression of components of the LPS-recognition complex remained unchanged, we provided evidences for alterations of the TLR4 signaling pathway in macrophages from E2-treated mice. Indeed, E2 treatment resulted in the inhibition of PI3K activity and Akt phosphorylation in LPS-activated macrophages, whereas NF-kappaB p65 transcriptional activity was concomitantly increased. Incubation of macrophages with the PI3K inhibitor wortmanin enhanced proinflammatory cytokine gene expression in response to TLR4 activation, and abolishes the difference between cells from placebo- or E2-treated mice, demonstrating the pivotal role of the PI3K/Akt pathway. We conclude that the macrophage activation status is enhanced in vivo by E2 through ERalpha and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.

By Homing to the Kidney, Activated Macrophages Potently Exacerbate Renal Injury

Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 x 10(6) to 5 x 10(6)) or activated (1 x 10(3) to 1 x 10(6)) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 x 10(4) macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 x 10(6) macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages.

Fibronectin Binding and Proteolytic Degradation byLeishmaniaand Effects on Macrophage Activation

Infection by vector-borne protozoa of the genus Leishmania occurs by the deposition of parasites within the skin of the mammalian host, where they eventually bind to and are phagocytized by Mphis. Our previous work supported the idea that parasites can interact with extracellular matrix and basement membrane proteins, such as fibronectin (FN), within the skin, leading to enhanced invasion. In this report, we extend these findings and show that both promastigotes and amastigotes of Leishmania species can bind directly to soluble FN and laminin (LM) and that promastigotes express a distinct surface protein of approximately 60 kDa that binds both FN and LM. Promastigotes of multiple Leishmania species can rapidly degrade FN by using surface-localized and secreted metalloprotease (leishmanolysin). FN degradation at the surfaces of amastigotes is leishmanolysin dependent, whereas both secreted leishmanolysin and cysteine protease B contribute to extracellular FN degradation. Leishmania-degraded FN decreased the production of reactive oxygen intermediates by parasite-infected macrophages and affected the accumulation of intracellular parasites. These findings show that both parasite stages of Leishmania species bind to and proteolytically degrade FN at the parasite surface and distantly through secreted proteases and that degraded forms of FN can influence the activation state of parasite-infected macrophages.

The Leishmania-macrophage interaction: a metabolic perspective

Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon sources and growth-limiting nutrients, respectively, and their availability to intracellular amastigotes may be regulated by the activation state of host macrophages. Metabolic processes in both the parasite and host cell may thus be crucial determinants of disease outcome.

Gas6 and Its Receptors Are Implicated in Sepsis as Modulators of Innate Immunity.

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P≤0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6−/− mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF-α was higher in Gas6−/− than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6−/− plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6−/− than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6−/− mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl−/−, Tyro3−/− and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6−/− mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6−/− macrophages produced more TNF-α and IL-6 than WT macrophages. Cytokine release by Gas6−/− macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6−/− macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6−/− macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NFκB activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6−/− mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NFκB activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6−/− mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.

Bioindicators of Immune Function in Creosote-adapted Estuarine Killifish, Fundulus heteroclitus

Several populations of the estuarine killifish, Fundulus heteroclitus, also known as the mummichog, exhibit characteristics of adaptation to priority pollutants. One such population of mummichog inhabits the Elizabeth River in Virginia at the Atlantic Wood site (AW), a U.S. Environmental Protection Agency (EPA) Superfund site heavily contaminated with creosote containing a mixture of polyaromatic hydrocarbons (PAHs). Although PAHs are known to be immunotoxic in experimental animals, resident AW mummichogs seem to thrive. Mummichogs from the AW site and a reference site were subsequently examined over a 2-yr period for total immunoglobin (IgM), as well as circulating antibody levels against 5 ubiquitous marine bacteria. Expression profiles of circulating and lymphoid lysozyme and lymphoid cyclooxygenase-2 (COX-2) were also examined. Compared to relatively high total IgM and specific antibody responses in reference fish, AW mummichogs had lower circulating IgM and lower specific antibody levels against all bacteria examined, however they had higher levels of circulating lysozyme. Lymphoid cells in the AW mummichogs also expressed higher levels of lysozyme, as well as COX-2, which may indicate a state of macrophage activation. Elevated COX-2 levels may be associated with enhanced metabolism of PAHs through cooxidation-peroxidase pathways. Additional studies attempted to immunize AW mummichogs reared in uncontaminated water to compare their antibody responses to that of reference fish. AW mummichogs did not survive 40 d post culture, while reference fish thrived. Our findings suggest that the chemical environment at the AW site may be vicariously enhancing components of innate immunity, probably through oxidative stress pathways, in resident mummichogs, while actively suppressing humoral immune responses.

Antigen presentation and transfer between B cells and macrophages

B cells play an active role in directing immunity against specific proteins in part because of their capacity to sequester antigen via B cell receptor (BCR). Our prior findings indicate that B cells can initiate an immune response in vivo to self proteins independent of other antigen-presenting cells (APC). However, these studies also demonstrated that both dendritic cells and macrophages are important in the ongoing immune response. The present work illustrates a mechanism by which antigen acquired by B cells through BCR is specifically transferred to other APC, in particular, macrophages. The transfer of antigen is dependent on the specificity of BCR and requires direct contact between the cells, but does not require MHC compatibility between the cells and is independent of the activation state of macrophages. Antigen transfer is functional, in that macrophages, which received B cell derived-antigen, can activate CD4 T cells. Overall, these results define a novel mechanism by which B cells can focus immunity toward a specific antigen and transfer the ability to activate CD4 T cells to other APC.

Protein Kinase Cζ (PKCζ) Regulates Ocular Inflammation and Apoptosis in Endotoxin-Induced Uveitis (EIU): Signaling Molecules Involved in EIU Resolution by PKCζ Inhibitor and Interleukin-13

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Czeta (PKCzeta). To understand the effect of interleukin-13 or PKCzeta inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-alpha (TNF-alpha) with nuclear factor kappaB (NF-kappaB)/p65 expression. Treatment led to absence of NF-kappaB/p65 expression and low levels of TNF-alpha, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-kappaB/p65 decreased and nuclear NF-kappaB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-alpha, indicating onset of spontaneous resolution. Treatment limited PKCzeta cleavage, with expression of nuclear NF-kappaB/p50 and BCL-3 and low nuclear NF-kappaB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCzeta by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-alpha, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-kappaB/p65, increased transforming growth factor-beta2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3.

Tumor Signaling to the Bone Marrow Changes the Phenotype of Monocytes and Pulmonary Macrophages during Urethane-Induced Primary Lung Tumorigenesis in A/J Mice

Little is known about how the composition of stromal cells within the lung cancer microenvironment varies during tumor progression. We examined by immunohistochemistry each of six different stromal cell populations during the development of chemically induced primary lung cancer in mice. Blood vessels were seen even in microscopic lesions, and their numbers increased with tumor size. Neutrophils infiltrated the alveoli of tumor-bearing lungs and within the periphery of macroscopic adenomas and adenocarcinomas. The numbers of peritumoral lymphocytes and macrophages increased during oncogeny, but quantitative changes in mast cells and fibroblasts were not evident. Because macrophage depletion reduces tumor growth and these cells are thus important to tumorigenesis, we also investigated their phenotype. Pulmonary macrophages expressed arginase I (subtype M2) but not inducible nitric-oxide synthase in lungs with premalignant lesions, whereas macrophages in carcinoma-bearing lungs expressed inducible nitric-oxide synthase (subtype M1) but not arginase I. Local pulmonary stimuli did not seem responsible for this shift in macrophage activation state because monocytes still residing within the bone marrow adopted these expression patterns before entering the circulation, presumably in response to tumor-derived signals. These biochemical markers of macrophage activation states would have diagnostic and/or therapeutic value if analogous systemic shifts occur in humans.

Induction of Golli-MBP Expression in CNS Macrophages During Acute LPS-Induced CNS Inflammation and Experimental Autoimmune Encephalomyelitis (EAE)

Microglia are the tissue macrophages of the CNS. Microglial activation coupled with macrophage infiltration is a common feature of many classic neurodegenerative disorders. The absence of cell-type specific markers has confounded and complicated the analysis of cell-type specific contributions toward the onset, progression, and remission of neurodegeneration. Molecular screens comparing gene expression in cultured microglia and macrophages identified Golli-myelin basic protein (MBP) as a candidate molecule enriched in peripheral macrophages. In situ hybridization analysis of LPS/IFNg and experimental autoimmune encephalomyelitis (EAE)–induced CNS inflammation revealed that only a subset of CNS macrophages express Golli-MBP. Interestingly, the location and morphology of Golli-MBP+ CNS macrophages differs between these two models of CNS inflammation. These data demonstrate the difficulties of extending in vitro observations to in vivo biology and concretely illustrate the complex heterogeneity of macrophage activation states present in region- and stage-specific phases of CNS inflammation. Taken altogether, these are consistent with the emerging picture that the phenotype of CNS macrophages is actively defined by their molecular interactions with the CNS microenvironment.

Role of Growth Arrest-Specific Gene 6 Product (Gas6) in Severe Sepsis.

Gas6 binding to its cognate receptor tyrosine kinases (Tyro3, Axl and Mer) down-regulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. Mer activation inhibits TNF-α production by macrophages and alleviates endotoxic shock in mice. Gas6 receptors are cleaved in the extracellular domain to generate soluble receptors. Soluble receptors function as decoy receptors, thereby inhibiting the Gas6 interaction with cell-associated receptors. The aim of the study was to determine whether Gas6 pathway plays a role in sepsis in human and mice. In the first part of the study, we measured plasma levels of Gas6 and its soluble receptors sTyro3 and sAxl in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with acute non-infectious inflammatory diseases. Gas6 and sAxl concentrations were higher in septic patients than in healthy subjects or in patients with non-infectious inflammatory disease (P≤ 0.0001 for Gas6 and <0.004 for sAxl) and correlated with C-reactive protein levels (P=0.0019 for Gas6 and =0.0037 for sAxl). The sensitivity and specificity of Gas6 levels to predict fatal outcome was 83% and 88%. In the second part of the study, we investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in Gas6+/+ and Gas6−/ − mice. Circulating levels of Gas6 after LPS 50mg/kg i.p. peaked at 1 h. (66±3ng/ml), were still elevated 4 h. later (47±1ng/ml) and returned approximately to baseline levels 8 h. after the LPS challenge (34±1ng/ml); P<0.001 for all time points versus baseline (23±1ng/ml). Cytokine (TNF-α and IL-6) production was higher in Gas6−/ − than Gas6+/+ mice after LPS 50mg/kg (TNF-α at 1 h.: 10±1ng/ml in Gas6−/ − versus 4.5±0.5ng/ml in Gas6+/+ mice, P<0.001; IL-6 at 4h.: 77±4ng/ml in Gas6+/+ versus 56±6ng/ml in Gas6−/ − mice, P=0.02). Similar data were obtained with Tyro3−/ − and Axl−/ − mice. Mortality induced by LPS 25mg/kg was 25% in Gas6+/+ versus 87% in Gas6−/ − mice (P=0.0023). In peritonitis models (cecal ligation and puncture, and i.p. injection of E. coli), plasma levels of Gas6 increased two-fold above baseline and remained elevated at least 24h. Increases of Gas6 levels were proportional to the importance of the size of the E. coli inoculum. In vitro experiments revealed that Gas6−/ − LPS-stimulated macrophages produced more TNF-α than Gas6+/+ macrophages. In conclusion, in septic patients, plasma levels of Gas6 and its cognate receptors were elevated and associated with fatal outcome. In mice, Gas6 plasma levels raised in experimental endotoxemia and sepsis models, and correlated also with sepsis severity. Thus, Gas6 and its receptors hold promise as early sepsis markers and outcome predictors, and could constitute therapeutic targets for new immunomodulating drugs.