Tissue-specific Macrophages Research Articles

Genetic Models of Macrophage Depletion.

Macrophages are a heterogeneous population of innate immune cells and are distributed in most adult tissues. Certain tissue-resident macrophages with a prenatal origin, together with postnatal monocyte-derived macrophages, serve as the host scavenger system to eliminate invading pathogens, malignant cells, senescent cells, dead cells, cellular debris, and other foreign substances. As a key member of the mononuclear phagocyte system, macrophages play essential roles in regulation of prenatal development, tissue homeostasis, and disease progression. Over the past two decades, considerable efforts have been made to generate genetic models of macrophage ablation in mice. These models support investigations of the precise functions of tissue-specific macrophages under physiological and pathological conditions. Herein, we overview the currently available mouse strains for in vivo genetic ablation of macrophages and discuss their respective advantages and limitations.

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature.

We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.

Specification of tissue-resident macrophages during organogenesis.

Tissue-resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro-myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine-receptor-dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.

Specific Neuropilins Expression in Alveolar Macrophages among Tissue-Specific Macrophages.

In the immune system, neuropilins (NRPs), including NRP-1 and NRP-2, are expressed in thymocytes, dendritic cells, regulatory T cells and macrophages. Their functions on immune cells around the neoplastic cells vary into pro-angiogenesis, tumor progression and anti-angiogenesis according to their ligands. Even though NRPs expression on malignant tumors and immune system has studied, a PubMed-based literature query did not yield any articles describing NRPs expression on tissue-specific macrophages. The aims of this study were (i) to detect NRPs expression on tissue-specific macrophages in the brain, liver, spleen, lymph node and lung; (ii) to observe NRPs expression in classes of macrophages, including alveolar macrophages (AMs), bronchial macrophages (BMs), interstitial macrophages (IMs), intravascular macrophages (IVMs) and macrophage subsets (M1, M2 and Mox) in lung; and (iii) to detect the co-expression of NRPs and dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in AMs. Both NRPs were specifically detected in AMs among tissue-specific macrophages by immunohistochemistry (IHC). NRPs mRNA expression levels were characterized in normal lung by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ-polymerase chain reaction (in situ-PCR). The expression of both NRPs was detected in AMs, BMs and IVMs by IHC. The frequency of NRPs+ AMs in lung tissue adjacent to the cancer margin was significantly higher than the frequencies in inflamed and normal lung tissue. Double and triple IHC demonstrated that NRPs are expressed on all macrophage subsets in lung. Double IHC showed co-expression of DC-SIGN and NRPs in AMs. This study demonstrated for the first time the specific expression of both NRPs in AMs among tissue-specific macrophages and their expression on M1, M2 and Mox macrophages. Furthermore, the possible origin of AMs from blood monocytes could be suggested from a co-expression of NRPs and DC-SIGN.

Transcriptional immunoresponse of tissue-specific macrophages in swine after infection with African swine fever virus

Abstract Macrophages and cytokines are important in the control of inflammation and regulation of the immune response. However, they can also contribute to immunopathology in the host after viral infection and the regulatory network can be subverted by infectious agents, including viruses, some of which produce cytokine analogues or have mechanisms that inhibit cytokine function. African swine fever virus (ASFV) encodes a number of proteins which modulate cytokine and chemokine induction, host transcription factor activation, stress responses, and apoptosis. The aim of this review is to elucidate the mechanisms of immune responses to ASFV in different subpopulations of porcine macrophages. A transcriptional immune response in different resident tissue macrophages following ASFV infection was presented in many publications. ASFV-susceptible porcine macrophages can be of several origins, such as peripheral blood, lungs, bone marrow, etc. blood monocytes, blood macrophages, and lung macrophages have demonstrated a modulation of phenotype. Monocyte-derived macrophages could express surface markers not found on their monocyte precursors. Moreover, they can undergo further differentiation after infection and during inflammation. When viruses infect such cells, immunological activity can be seriously impaired or modified.

Local up-regulation of interferon-γ (IFN-γ) following disc herniation is involved in the inflammatory response underlying acute lumbar radicular pain

Abstract Aims Lumbar radicular pain after disc herniation may be associated release of pro-inflammatory cytokines from nucleus pulposus (NP) tissue. In the present study we examined the role of interferon-γ (IFN-γ) and cluster of differentiation 68 (CD68) in the acute phase of this process. Methods First, in an animal model mimicking the clinical situation after disc herniation, the role of IFN-γ on the dorsal horn single cell activity and gene expression close to the nerve roots was studied. Second, in patients with severe lumbar radicular pain due to disc herniation, we examined how two single nucleotide polymorphisms (SNPs; rs2069705 and rs2069718) important for the IFN-γ expression influenced the pain and disability measured by visual analogue scale (VAS) and Oswestry Disability Index (ODI). Results The animal data demonstrated a significant increase in the nociceptive activity at the spinal level after local application of NP and IFN-γ onto the nerve-roots. A positive correlation between IFN-γ and CD68 in the NP tissue was also observed. Moreover, the data of the patients revealed that carriers of the IFN-γ SNPs; rs2069705 A allele and rs2069718 G allele had an increased disability score i.e. ODI. Conclusions The present data suggest that IFN-γ through activation of tissue-specific macrophages close to the nerve roots may be important for acute inflammatory pain and disability following lumbar disc herniation.

Identification of a Tissue-Specific, C/EBPβ-Dependent Pathway of Differentiation for Murine Peritoneal Macrophages

Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-β. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPβ-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPβ plays an intrinsic, tissue-restricted role in the generation of resident macrophages.

Hypoxia: how does the monocyte-macrophage system respond to changes in oxygen availability?

Hypoxia is an important feature of inflamed tissue, such as the RA joint. Activated monocytes/macrophages and endothelial cells play a pivotal role in the pathogenesis of RA, implicated in the mechanism of inflammation and erosion. During development, myeloid progenitor cells sequentially give rise to monoblasts, promonocytes, and monocytes that are released from the bone marrow into the bloodstream. After extravasation, monocytes differentiate into long-lived, tissue-specific macrophages or DCs. The effect of different oxygen concentrations experienced by these cells during maturation represents a novel aspect of this developmental process. In inflamed joint tissue, the microvascular architecture is highly dysregulated; thus, efficiency of oxygen supply to the synovium is poor. Therefore, invading cells must adapt instantaneously to changes in the oxygen level of the microenvironment. Angiogenesis is an early event in the inflammatory joint, which is important in enabling activated monocytes to enter via endothelial cells by active recruitment to expand the synovium into a "pannus", resulting in cartilage degradation and bone destruction. The increased metabolic turnover of the expanding synovial pannus outpaces the dysfunctional vascular supply, resulting in hypoxia. The abnormal bioenergetics of the microenvironment further promotes synovial cell invasiveness. In RA, joint hypoxia represents a potential threat to cell function and survival. Notably, oxygen availability is a crucial parameter in the cellular energy metabolism, itself an important factor in determining the function of immune cells.

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7. Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8. For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

Highlights from the Literature

Highlights from the Literature

Macrophages in inflammation and its resolution

Macrophages are highly plastic leukocytes that differentiate from monocytes following their entry into extravascular tissues. Macrophages can enter various tissues under inflammatory or non-inflammatory conditions and assume different functions and phenotypes according to the cues they receive from the environment. The notion that inflammation in general and macrophage responses in particular affect physiological phenomena that were previously considered to be not immune-related has enhanced and broadened our understanding of macrophage function during inflammation and its resolution. This volume brings together 14 manuscripts that cover various aspects of macrophage function during inflammation and its resolution, as well as in several pathologic states for which a significant, long-lasting, macrophage-mediated immune response plays a significant role. Two of the manuscripts present original research on macrophage phagocytosis and its implications. Five provide an overview of macrophage function during inflammation and its resolution, with an emphasis on the modulatory role of particular elements in this response, such as apoptotic leukocytes, specific pathogens, hypoxia, and hormone receptors. The remaining seven manuscripts outline the role of macrophages during inflammation and its resolution in different tissues, including the lung, cardiovascular and adipose tissues, injured skeletal muscle and neuronal tissues, and synovial and oral cavities. The two original research articles are devoted to the consequences of particle engulfment by macrophages. Labrousse et al. (2011) describe a novel experimental strategy in which they use micro-patterned immune complexes to trigger frustrated phagocytosis and thereby determine spatial parameters in lysosomal movement and fusion. Janko et al. (2011) report on the cumulative binding of CRP and anti-CRP antibodies to the surface of secondary necrotic cells. This binding leads to a pro-inflammatory cytokine response following engulfment by macrophages, implying a potential role for these elements in the etiology of systemic lupus erythematosus. Of the review articles that discuss the regulation of macrophage differentiation and function by discrete events, two cover the interaction between macrophages and apoptotic leukocytes during the resolution of inflammation. Korns et al. (2011) outline the regulation of apoptotic cell clearance by macrophages (efferocytosis) and the environmental cues that promote the efferocytic capabilities of macrophages. The second manuscript by Ariel and Serhan (2012) reviews the impact of apoptotic cell sensing and disposal by macrophages on the switches in functional phenotypes displayed by these cells. The effect of another environmental factor, hypoxia, on monocyte/macrophage activation, and differentiation through transcriptional and translational modulation is covered by Rahat et al. (2011). Lugo-Villarino et al. (2011) discuss the pathogenesis and co-mortality displayed by two macrophage-inhabiting microbes (HIV and Mycobacterium Tuberculosis) and their influence on macrophage polarization. Patel et al. (2011) review the role of melanocortin receptor expression by macrophages in anti-inflammation and the resolution of inflammation, with attention given to melanocortin receptor agonists as therapeutic agents. Several review articles discuss the function of macrophages during inflammation and/or its resolution within distinct anatomical sites, taking into account the unique features of these tissue-specific macrophages, in particular the distinct environments in which they reside and their interactions with neighboring cells. Claria et al. (2011) review current knowledge on the contribution of macrophages to the inflammatory state characterizing adipose tissue and the phenotypic changes observed in macrophages during obesity. Kennedy et al. (2011) discuss macrophage polarization occurring within the synovial space of arthritic joints and its modulation by cytokines, transcription factors, and pro-resolving lipid mediators. The article from Bosurgi et al. (2011) describes the multiple actions of macrophages in injured skeletal muscle, where the effects of these cells are a double-edged sword and can either promote healing and repair or lead to fibrosis and fat replacement. Herold et al. (2011) survey the indispensable role of macrophages in the resolution and termination of inflammation in lung infection and injury as well as the molecular pathways involved in these processes. Proper termination of inflammatory events and clearance of apoptotic cells are also critical to the cardiovascular system, as reviewed by Thorp (2012), and defects in macrophage efferocytosis can lead to atherosclerosis and myocardial infarction. While monocyte-derived macrophages and resident microglia were previously considered to be detrimental in brain inflammation and injury, recent advances reviewed by Jung and Schwartz (2012) suggest an opposite role for these macrophage-like cells, with a positive impact on brain maintenance and repair. Finally, Hasturk et al. (2012) outline the reciprocal interaction between periodontal disease and chronic inflammatory illnesses and the role that macrophages play in mediating these chronic inflammatory diseases. Altogether, the articles in this volume portray the complexity of the multiple roles played by macrophages and members of their lineage during inflammation and its resolution, and their manipulation by the injured milieu. These topics are currently heavily studied, and advances in the field, facilitated by state-of-the-art genetics and optical technologies, will undoubtedly continue to contribute to our understanding of the immune system's response to foreign insults, trauma, and inflammatory disorders.

C/EBPβ mediates the differentiation of peritoneal, but not splenic or liver, tissue macrophages (153.28)

Abstract Macrophages are dispersed throughout the body but exhibit tissue-specific phenotypes and functions. The extracellular and transcriptional pathways that regulate the generation of tissue-specific macrophages are largely unknown. In mice, the peritoneal cavity contains two distinct populations of macrophages, small (CD11b+F4/80lowCD93-) and large (CD11b+F4/80hiCD93+) peritoneal macrophages (SPM and LPM, respectively). Here, we show that mice deficient for the transcription factor C/EBPβ have increased numbers of SPM compared to control littermates, but lack LPM. In C/EBPβ-sufficient and deficient mice, however, splenic (MOMA-1+ and F4/80+) and liver (F4/80+) macrophage compartments are equivalent. When adoptively transferred into C/EBPβ-/- mice, wild-type SPM give rise to LPM, revealing a differentiative pathway from SPM to LPM that requires C/EBPβ. A role for C/EBPβ in the differentiation of certain macrophages is also evident in vitro, as bone marrow cells from C/EBPβ-sufficient and -deficient mice produce macrophages equivalently in cultures containing M-CSF, but C/EBPβ-/- bone marrow cells fail to generate normal macrophages in GM-CSF cultures. These findings demonstrate that tissue-specific macrophages arise through distinct transcriptional pathways, and reveal C/EBPβ as a master regulator of differentiation for a restricted subset of tissue macrophages.

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.

Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells

Carriage of the TNF -308 A allele (rs1800629 A) has been associated with increased serum TNF-alpha levels, the development of sepsis syndrome, and fatal outcome, in severely traumatized patients (Menges et al., 2008 [1]). Herein, we analysed the putative allelic imbalance of TNF-alpha release from myeloid cells. Circulating peripheral blood cells from healthy human blood donors (n=104) and monocyte-derived macrophages (n=158) were analysed for their ex vivo capacity of TNF-alpha expression. Our findings indicate that carriage of the TNF -308 A allele is not associated with high TNF-alpha expression in circulating human leucocytes and monocyte-derived macrophages. Other cellular sources, e.g. tissue-resident cells like mast cells and/or tissue specific macrophages might be the cellular source of high TNF-alpha serum levels shortly after trauma.

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

The Roles of Endogenously-produced Chemokines in the Development of Tissue-specific Myeloid-lineage Cells and Macrophages

Chemokines play roles not only in chemotaxis of immune cells but also in the development and healing of tissue. Each tissue has its intrinsic myeloid lineage cells for maintenance of the specific tissue, such as microglia in brain, kupffer cells in liver and osteoclasts in bone.

Chlamydia and apoptosis: life and death decisions of an intracellular pathogen.

The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints. The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages. One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells. The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others. This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival - even under the most adverse circumstances.

Proliferation of naive T lymphocytes and T lymphoblasts in the presence of tissue specific macrophages

In this study the antigen-presenting ability of tissue specific macrophages isolated from bone marrow, spleen, peritoneal cavity and lungs was analyzed. Murine macrophages were isolated by a one-step adherence procedure (for 24 hours) and pretreated with mytomycin C. The antigen-presenting ability of the macrophages was tested in T cell proliferation assays. The ability of macrophages to support antigenspecific proliferation of T lymphoblasts was investigated when sheep red blood cell (SRBC)-specific T blasts were stimulated in vitro by antigen in the presence of different numbers of tissue specific macrophages. On the other hand, the abilities of macrophages to induce proliferation of naïve T cells were analyzed in allogeneic and syngeneic mixed leukocyte reactions (MLRs). It was demonstrated that tissue specific macrophages supported antigen specific proliferation of T lymphoblasts in vitro. They also induced the activation of allogeneic and syngeneic T cells. Increasing the number of macrophages co-cultured with T cells, led to a certain inhibitory effect on T cell proliferation.

Properties of tissue specific macrophages before and after in vitro activation

Macrophages derived from different tissues: bone marrow, spleen, peritoneal cavity and alveolus, were examined from the aspects of their morphology and functional characteristics expression of Fc receptors (FcR), phagocytic activity towards yeast particles and nonspecific esterase (NSE) content¹ before and after in vitro activation. Twenty four-hour-adherent cells were isolated with the aim of analyzing the characteristics of resident tissue macrophages. Following cultivation in vitro 8-day-adherent cells were used to investigate the influence of macrophage activation on their morphology and function. Morphological analysis of cell smears, performed in respect to cell size, showed significant enlargement, especially in the population of alveolar cells cultured for 8 days and activated with colony-stimulating factors (CSFs) and lymphokines. It was also demonstrated that 24-hour- and 8-dayadherent macrophages derived from different tissues exhibited similar properties. All these cells were more than 90% FcR-positive (FcR+) NSE-positive (NSE+) and had phagocytic properties. However, within the population of alveolar macrophages there were some NSE+ cells lacking FcR and phagocytic activity, even after in vitro activation. These results confirmed that the properties of alveolar macrophages differing from those of macrophages from other tissues were dependent on their microenvironment.