Recruitment Of Myelomonocytic Cells Research Articles

Macrophages as tools and targets in cancer therapy.

Tumour-associated macrophages are an essential component of the tumour microenvironment and have a role in the orchestration of angiogenesis, extracellular matrix remodelling, cancer cell proliferation, metastasis and immunosuppression, as well as in resistance to chemotherapeutic agents and checkpoint blockade immunotherapy. Conversely, when appropriately activated, macrophages can mediate phagocytosis of cancer cells and cytotoxic tumour killing, and engage in effective bidirectional interactions with components of the innate and adaptive immune system. Therefore, they have emerged as therapeutic targets in cancer therapy. Macrophage-targeting strategies include inhibitors of cytokines and chemokines involved in the recruitment and polarization of tumour-promoting myeloid cells as well as activators of their antitumorigenic and immunostimulating functions. Early clinical trials suggest that targeting negative regulators (checkpoints) of myeloid cell function indeed has antitumor potential. Finally, given the continuous recruitment of myelomonocytic cells into tumour tissues, macrophages are candidates for cell therapy with the development of chimeric antigen receptor effector cells. Macrophage-centred therapeutic strategies have the potential to complement, and synergize with, currently available tools in the oncology armamentarium.

Glia limitans superficialis oxidation and breakdown promote cortical cell death after repetitive head injury.

Repetitive mild traumatic brain injuries (mTBI) disrupt CNS barriers, the erosion of which has been linked to long-term neurodegenerative and psychiatric conditions. Although much attention has been devoted to CNS vasculature following mTBI, little is known about the glia limitans superficialis — a barrier of surface-associated astrocytes that helps protect the CNS parenchyma and maintain homeostasis. Here, we identify the glia limitans superficialis as a crucial barrier surface whose breakdown after acute repeat mTBI facilitates increased cell death and recruitment of peripheral myelomonocytic cells. Using intravital microscopy, we show that brain-resident microglia fortify this structure after a single mTBI, yet they fail to do so following secondary injury, which triggers massive recruitment of myelomonocytic cells from the periphery that contribute to further destruction of the glia limitans superficialis but not cortical cell death. We demonstrate, instead, that reactive oxygen species (ROS) generated in response to repetitive head injury are largely responsible for enhanced cortical cell death, and therapeutic administration of the antioxidant glutathione markedly reduces this cell death, preserves the glia limitans, and prevents myelomonocytic cells from entering the brain parenchyma. Collectively, our findings underscore the importance of preserving the glia limitans superficialis after brain injury and offer a therapeutic means to protect this structure and the underlying cortex.

Adrenomedullin promotes the growth of pancreatic ductal adenocarcinoma through recruitment of myelomonocytic cells.

Stromal infiltration of myelomonocytic cells is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and is related to a poor prognosis. However, the detailed mechanism for the recruitment of myelomonocytic cells to pancreatic cancer tissue remains unclear. In the present study, pancreatic cancer cells secreted high levels of adrenomedullin (ADM), and CD11b+ myelomonocytic cells expressed all components of ADM receptors, including GPR182, CRLR, RAMP2 and RAMP3. ADM enhanced the migration and invasion of myelomonocytic cells through activation of the MAPK, PI3K/Akt and eNOS signaling pathways, as well as the expression and activity of MMP-2. ADM also promoted the adhesion and trans-endothelial migration of myelomonocytic cells by increasing expression of VCAM-1 and ICAM-1 in endothelial cells. In addition, ADM induced macrophages and myeloid-derived suppressor cells (MDSCs) to express pro-tumor phenotypes. ADM knockdown in tumor-bearing mice or administration of AMA, an ADM antagonist, significantly inhibited the recruitment of myelomonocytic cells and tumor angiogenesis. Moreover, in vivo depletion of myelomonocytic cells using clodronate liposomes suppressed the progression of PDAC. These results reveal a novel function of ADM in PDAC, and suggest ADM is a promising target in the treatment of PDAC.

Inflammasome-Mediated Inhibition of Listeria monocytogenes-Stimulated Immunity Is Independent of Myelomonocytic Function

Activation of the Nlrc4 inflammasome results in the secretion of IL-1β and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8+ T cell responses than wt L. monocytogenes. It is also known that IL-1β orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24–48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8+ T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8α+ DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8α+ DCs, which are known to be critical for T cell response to L. monocytogenes.

Treatment with anti-CR3 antibodies ED7 and ED8 suppresses experimental allergic encephalomyelitis in Lewis rats.

Experimental allergic encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS). Among the leukocytes which infiltrate the CNS during EAE, numerous macrophages are present. These macrophages are thought to play a crucial role in the generation of tissue damage and attendant neurological deficits. The mechanism by which the macrophages migrate across the blood-brain barrier is not yet clear. Membrane proteins involved in macrophage adherence to the endothelium include the CD11b/CD18 integrin, also known as the type 3 complement receptor (CR3). In this study we show that two monoclonal antibodies (mAb) ED7 and ED8 are directed against rat CR3. In addition, these mAb reduce recruitment of myelomonocytic cells towards thioglycollate induced peritonitis by 15-33%. This indicates that both ED7 and ED8 interfere with an epitope on CR3, which is involved in recruitment of phagocytes towards inflammatory lesions. Intravenous injection of ED7 and ED8 suppressed clinical signs of EAE. MRC OX-42, which also recognizes CR3, did not reduce thioglycollate-induced phagocyte recruitment into the peritoneum, and had no effect on EAE. These findings suggest that CR3 plays a role in the recruitment of macrophages towards the inflamed CNS of EAE animals, and confirm the role of macrophages in the generation of clinical signs of EAE. Involvement of CR3 in other phagocyte immune functions during EAE is discussed.

Protective immunity and granuloma formation are mediated by two distinct tumor necrosis factor alpha- and gamma interferon-dependent T cell-phagocyte interactions in murine listeriosis: dissociation on the basis of phagocyte adhesion mechanisms.

Listeria-immune mice are able to express protective immunity in the absence of CD4+ T cells and an apparent granulomatous inflammation. Using a monoclonal antibody (5C6) able to inhibit the recruitment of myelomonocytic cells into inflammatory foci by binding to complement receptor type 3 (CR3/CD11b), we could show that protective immunity and granuloma formation indeed depend on two distinct types of T cell-phagocyte interactions. Listeria-specific CD8+ T lymphocytes, possibly in collaboration with CD4- CD8- T cells, rapidly interact with myelomonocytic cells infiltrating infected tissues in a CR3/CD11b-dependent manner. This interaction results in potent antilisterial protection but not in granuloma formation. On the contrary, CD4+ T cells are able to induce adhesion mechanisms that allow the accumulation of monocytes in granulomatous lesions even in the presence of monoclonal antibody 5C6. However, the protective capacity of these CR3/CD11b-independent T cell-mediated immune mechanisms is low in listeriosis. Tumor necrosis factor alpha and gamma interferon, known to be essential for the expression of both resistance and acquired immunity, are shown to be necessarily involved in granuloma formation, too. It therefore remains to be explained why CD8+ T cells, able to secrete both cytokines, do not induce granuloma formation. The data point to the presence of an as yet undefined CD4+ T cell-derived granuloma-inducing factor and favor the hypothesis that CD8+ T cells, in collaboration with circulating phagocytes, mediate immunity by rapidly liberating listeriae from permissive cells or protecting them from becoming infected.

Dynamics of lymphocytes and inflammatory cells recruited in liver during murine listeriosis. A cytofluorimetric study.

During primary infection of mice by Listeria monocytogenes, bacterial elimination is dependent on the recruitment of myelomonocytic cells in the infectious foci and the activation of their bactericidal mechanisms through cytokines secreted by Listeria-specific T lymphocytes. The immune events occurring in the liver, one of the main infected organs, have not yet been studied in detail. In the present quantitative study, we describe the dynamics of recruitment of cells belonging to the lymphoid or myelomonocytic lineages in the liver. The different cell populations mobilized into the liver were isolated each day during the course of a sublethal L. monocytogenes infection and their phenotype was characterized by flow cytometry. Three distinct phases of recruitment were observed. 1) During the first day of infection, 17 x 10(6) lymphomyeloid cells were recruited in the liver with a predominance of myelomonocytic cells; 51% of the incoming cells were M1/70+; the NK cell population (detected by the 4D11 antibody) also increased transiently at this period. 2) From day 3 to 5, a high number of myelomonocytic cells infiltrated the liver (13 x 10(6) M1/70+ cells); most of these cells were macrophages (as detected with the macrophage-restricted antibody FA/11 or observed after May-Grünwald Giemsa staining); the antigranulocytic antibodies 7/4 and RB6.8C5 were found to label these mononuclear phagocytes at this period of infection. A subpopulation of Thy-1+ cells (16%) was found to be labeled by the RB6.8C5 antibody in normal liver and, at day 5 and 6, all Thy-1+ cells also bound the RB6.8C5 mAb.3) From day 5 onward, two waves of phenotypically distinct T lymphocytes were observed; the number of CD8+ T lymphocytes (15 x 10(6) cells) increased first at day 5 and peaked on day 7; CD4+ T lymphocytes (6.2 x 10(6) cells) were then recruited with a 2-day delay (on day 7) in the liver.

Role of CR3 in Induced Myelomonocytic Recruitment: Insights From In Vivo Monoclonal Antibody Studies in the Mouse

In this minireview, I have therefore concentrated upon insights gained into the role of CR3 in the recruitment of myelomonocytic cells to inflammatory sites in the mouse, one small area of current in vivo experimentation manipulating members of the CD18 family of leukocyte adhesion molecules

The Role of the Type 3 Complement Receptor in the Induced Recruitment of Myelomonocytic Cells to Inflammatory Sites in the Mouse

The type 3 complement receptor (CR3), initially identified as the leukocyte cell surface receptor for iC3b, is now known to form part of the extended integrin family of cell adhesion molecules that mediate both cell-cell and cell-extracellular matrix interactions. The identification of a heritable deficiency of human leukocyte adhesion together with the advent of monoclonal antibodies has shed some light on the central role of CR3 in the transendothelial migration of macrophages and neutrophils to sites of inflammation. We review the general structural features of CR3 and then examine our understanding of its role in both nonspecific and T cell-dependent inflammatory processes based on our murine in vivo experiments. CR3-dependent inflammation seems to contribute to the pulmonary response to some stimuli (lipopolysaccharide) but not to others (bacillus Calmette-Guerin). These studies highlight the potential therapeutic benefits, as well as the significant risks of potentiating acute bacterial infections, of CR3 blockade in vivo.

Synergy between activated Leishmania major-specific CD4 + T lymphocytes and bone-marrow-derived cells in the exacerbation of murine cutaneous leishmaniasis

Mechanisms of exacerbation of murine cutaneous leishmaniasis mediated by Leishmania major-specific CD4 + T lymphocytes were studied. Using a limiting dilution assay for the quantification of Leishmania parasites, the infected tissues (footpad) of lethally irradiated mice were found to contain tenfold less parasites at four days of infection than the footpads of infected unirradiated animals. Injection of bone marrow cells depleted of T cells into irradiated mice at the site of infection led to an increase in parasite numbers to levels equivalent to those seen in unirradiated mice. After injection of either L. major-specific CD4 + T cells, previously shown to exacerbate cutaneous leishmaniasis, into the infected footpad or the intravenous (i.v.) injection of bone marrow cells depleted of T cells, the numbers of parasites in lesions of irradiated mice never reached the values found in unirradiated control mice. In contrast, the concomitant transfer of CD4 + T-cell populations in situ and bone marrow cells depleted of T cells intravenously led to an increase in parasite loads in irradiated mice up to levels comparable to those of the unirradiated mice. This suggested that recruitment of myelomonocytic cells at the site of the lesions plays a role in the exacerbation of murine cutaneous leishmaniasis mediated by these CD4 + T lymphocytes. Finally, a similar effect was observed with T cells specific for an antigen unrelated to Leishmania, provided that this antigen was added to the L. major infecting inoculum.

Synergy between activated Leishmania major-specific CD4 + T lymphocytes and bone-marrow-derived cells in the exacerbation of murine cutaneous leishmaniasis

Mechanisms of exaberbation of murine cutaneous leishmaniasis mediated by Leishmania major-specific CD4 + T lymphocytes were studied. Using a limiting dilution assay for the quantification of Leishmania parasites, the infected tissues (footpad) of lethally irradiated mice were found to contain tenfold less parasites at four days of infection than the footpads of infected unirradiated animals. Injection of bone marrow cells depleted of T cells into irradiated mice at the site of infection led to an increase in parasite numbers to levels equivalent to those seen in unirradiated mice. After injection of either L. major-specific CD4 + T cells, previously shown to exacerbate cutaneous leishmaniasis, into the infected footpad or the intravenous (i.v.) injection of bone marrow cells depleted of T cells, the numbers of parasites in lesions of irradiated mice never reached the values found in unirradiated control mice. In contrast, the concomitant transfer of CD4 + T-cell populations in situ and bone marrow cells depleted of T cells intravenously led to an increase in parasite loads in irradiated mice up to levels comparable to those of the unirradiated mice. This suggested that recruitment of myelomonocytic cells at the site of the lesions plays a role in the exacerbation of murine cutaneous leishmaniasis mediated by these CD4 + T lymphocytes. Finally, a similar effect was observed with T cells specific for an antigen unrelated to Leishmania, provided that this antigen was added to the L. major infecting inoculum.

Antibody to the murine type 3 complement receptor inhibits T lymphocyte-dependent recruitment of myelomonocytic cells in vivo.

We have used the delayed-type hypersensitivity (DTH) response to SRBC or tuberculin to examine the role of the murine type 3 complement receptor in T lymphocyte-dependent inflammatory recruitment. Intravenous injection of 5C6, a CR3-specific rat mAb known to impair myelomonocytic adhesion, divided the DTH to SRBC in actively immunized mice into two phases. The early phase, which lasted 24 h, was characterized by maximal oedema and maximal inflammatory recruitment and was 5C6 inhibitable. The later phase was 5C6 resistant and reached a peak 48 h after antigenic challenge and was superimposable on the declining peak seen in control mice. Passive transfer of reactive T cells mixed with antigen was used to examine the myelomonocytic effector arm of the DTH alone. Both passive transfer of cutaneous DTH to SRBC and passive transfer of the largely monocytic T cell-dependent recruitment to tuberculin in the peritoneal cavity were completely abolished by systemic 5C6 treatment. Injection of 5C6-treated donor leukocytes at the site of passive transfer had no effect. Treatment of donor mice with 5C6 at the time of active immunization did not alter their ability to provide reactive T cells for passive transfer. The myelomonocyte-restricted rat mAb 7/4 and the rapidly cleared F(ab')2 fragment of 5C6 showed no inhibition of the DTH. In all cases, inhibition of footpad swelling correlated with histological evidence of inhibition of myelomonocytic cell recruitment. Peritoneal cell counts after local DTH to tuberculin showed complete inhibition of monocyte recruitment. We conclude that CR3 plays a quantitatively important role in T cell-dependent inflammatory recruitment. This is absolute in passive transfer experiments, but only partial after active immunization. Leukocyte CR3 plays a common role in both immunologically specific and nonspecific inflammatory recruitment and provides a target that could possibly be manipulated to therapeutic advantage.

Absence of Wallerian Degeneration does not Hinder Regeneration in Peripheral Nerve.

Wallerian degeneration of the distal stump of a severed peripheral nerve involves invasion by myelomonocytic cells, whose presence is necessary for destruction of myelin and for initiating mitosis in Schwann cells (Beuche and Friede, 1984). Degeneration of the distal ends of the axons themselves is assumed to occur by autolytic mechanisms. We describe a strain of mice (C57BL/6/Ola) in which leucocyte invasion is slow and sparse. In these mice, confirming Beuche and Friede, myelin removal is extremely slow. A new finding is that axon degeneration is also very slow. This is a consequence of lack of recruitment of myelomonocytic cells for if such recruitment is prevented in other mouse strains by a monoclonal antibody against the complement type 3 receptor (Rosen and Gordon, 1987) axon degeneration is again slowed. We have also, surprisingly, found that nerve regeneration in the C57BL/6/Ola mice is not impeded by the presence of largely intact axons in the distal stump and absence of recruited cells, myelin debris and the absence of Schwann cell mitosis.

Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo.

Macrophage interactions with extracellular matrix and other cells are important in phagocytosis, inflammation, and immunity. To learn more about the surface molecules involved in adhesion we compared the binding of murine macrophages and polymorphonuclear leukocytes (PMN) with artificial substrate in vitro. A distinctive type of adhesion of thioglycollate-elicited peritoneal macrophages (TPM) to bacteriologic plastic (BP) was defined, which was pronase-sensitive, Mg2+-dependent, and required cytoskeletal stabilization. A rat mAb designated 5C6 was isolated because it inhibited TPM attachment to BP, as well as mediating detachment of TPM adherent to that substratum. In addition, it inhibited the attachment of PMN to tissue culture plastic. This antiadhesive property of 5C6 mAb required intact IgG; the F(ab')2 fragment was partially effective and Fab was ineffective. 5C6 recognized the type 3 complement receptor, inhibiting rosetting of EAC3bi to TPM and immunoprecipitating a heterodimer of 160 and 95 kD that comigrated with the M1/70 immunoprecipitate. 5C6 recognized a pronase-stable epitope distinct from that of M1/70. Other mAbs, including M1/70 (CR3) and 2.4G2 (FcR), failed to have any antiadhesive effect in vitro. The inhibitory activity of 5C6 in short-term adhesion assays correlated with its inhibition of recruitment of myelomonocytic cells to a thioglycollate-elicited peritoneal exudate in vivo, after intravenous injection of mAb. 5C6 IgG inhibited recruitment of myelomonocytic cells by 84 +/- 3% at 1 d compared with saline-injected controls. The F(ab')2 fragment and a class-matched control IgG had little effect. Recruitment of TPM at 4 d was also efficiently inhibited by 5C6 IgG. 5C6 IgG was not cytotoxic, had no effect on marrow egress, did not cause increased phagocytic clearance of circulating neutrophils, and had no adverse effect on chemotaxis in vitro. We show that CR3 alone of the LFA-family is necessary for the recruitment of myelomonocytic cells to inflammatory stimuli such as thioglycollate broth. This strategy may be of general use in isolating reagents that inhibit the adhesive function of CR3 and provides a novel approach to antiinflammatory therapy.