F4/80 and the related adhesion-GPCRs

The F4/80 monoclonal antibody was first reported in this journal 30 years ago (Eur. J. Immunol. 1981. 11: 805-815). F4/80 has become a widely used marker for monocytes and many, but not all, tissue macrophages in the mouse. F4/80 is a member of the EGF-TM7 family of leukocyte plasma membrane heptahelical molecules, which includes CD97 and EMR2. This Viewpoint summarises current knowledge of the expression, structure and functions of the EGF-TM7 family, as part of a larger family of tissue adhesion-GPCRs.     

Inactivation of the EGF-TM7 receptor EMR4 after the Pan-Homo divergence

We here report on the identification of a novel human EGF-TM7 receptor, designated EMR4. Like most EGF-TM7 receptor genes, EMR4 is localized on the short arm of chromosome 19, in close proximity to EMR1. Remarkably, due to a one-nucleotide deletion in exon 8, translation of human EMR4 would result in a truncated 232-amino acid protein lacking the entire seven-span transmembrane region. This deletion is not present in nonhuman primates, including chimpanzees, suggesting that EMR4 became nonfunctional only after human speciation, about five million years ago. Thus, EMR4 surprisingly accounts for a genetic difference between humans and primates related to immunity.     

The macrophage cell surface glycoprotein F4/80 is a highly glycosylated proteoglycan

Molecules whose expression is limited to particular leukocyte populations are of interest since they may perform unique functions for these cells. We therefore examined the biochemical nature of the F4/80 molecule, which is expressed solely on macrophage and dendritic cell subpopulations. Our study clearly indicates that post-translational modifications, which can influence both a protein's structural and functional features, constitute a major component of the 160-kDa cell-surface F4/80 molecule. The F4/80 molecule is synthesized as a single polypeptide chain which acquires numerous intramolecular disulfide bonds and requires an extended time period (T_1/2 = 60 min) for transport to an endoglycosidase H-resistant form. The F4/80 molecule contains extensive N-linked glycosylation which contributes approximately 40 kDa to the mature molecule. The N-linked carbohydrates are of the branched, complex type, containing repeating N-acetylglycosamine or N-acetyllactosamine units which mediate the reactivity of the F4/80 molecule with Datura stramonium lectin. O-linked glycosylation is also present and contributes approximately 10 kDa to the F4/80 molecule. Furthermore, the sialic acid modifications of the F4/80 molecule are primarily through α2-6 linkages to galactose. Finally, we demonstrate that the F4/80 molecule is a proteoglycan modified by chondroitin sulfate glycosaminoglycans. In addition to clarifying the nature of the F4/80 molecule biochemically, these posttranslational modifications have specific implications for molecular recognition processes. We conclude that the modifications of the F4/80 molecule may mediate cell-cell recognition, cell adhesion, or ligand binding independently of the F4/80 molecule protein core.     

The chemokine receptor CCR6 is an important component of the innate immune response

In our initial studies we found that naïve CCR6‐deficient (CCR6^–/–) C57BL/6 mice possessed significantly lower number of both F4/80⁺ macrophages and dendritic cells (DC), but higher number of B cells in the peritoneal cavity, as compared to naïve wild type (WT) controls. Furthermore, peritoneal macrophages isolated from CCR6^–/– mice expressed significantly lower levels of inflammatory cytokines and nitric oxide following lipopolysaccharide (LPS)stimulation, as compared to WT macrophages. In a severe experimental peritonitis model induced by cecal ligation and puncture (CLP), CCR6^–/– mice were protected when compared with WT controls. At 24 h following the induction of peritonitis, CCR6^–/– mice exhibited significantly lower levels of inflammatory cytokines/chemokines in both the peritoneal cavity and blood. Interestingly, DC recruitment into the peritoneal cavity was impaired in CCR6^–/– mice during the evolution of CLP‐induced peritonitis. Peritoneal macrophages isolated from surviving CCR6^–/– mice 3 days after CLP‐induced peritonitis exhibited an enhanced LPS response compared with similarly treated WT peritoneal macrophages. These data illustrate that CCR6 deficiency alters the innate response via attenuating the hyperactive local and systemic inflammatory response during CLP‐induced peritonitis.     

Expression of IL-1Rrp2 by human myelomonocytic cells is unique to DCs and facilitates DC maturation by IL-1F8 and IL-1F9

We report for the first time that expression of the novel IL-1 cytokine receptor IL-1Rrp2 (IL-1R6) is unique to DCs within the human myelomonocytic lineage. IL-1Rrp2 was expressed by monocyte-derived dendritic cells (MDDCs) which was dose-dependently increased by IL-4 and correlated with increased numbers of differentiated MDDCs. Human plasmacytoid DCs also express IL-1Rrp2 but the receptor is not expressed by either myeloid DC type 1 (mDC1) or mDC2 cells. We also show that IL-1F8 or IL-1F9 cytokines, which signal through IL-1Rrp2, induce maturation of MDDCs, as measured by increased expression of HLA-DR and CD83 and decreased expression of CD1a. Furthermore, IL-1F8 stimulated increased CD40 and CD80 expression and IL-18 and IL-12 p70 production by MDDCs, which induced proliferation of IFN-γ-producing CD3(+) lymphocytes (indicative of inflammatory Th1 subsets). IL-1F8 and IL-1F2 were equipotent in their ability to stimulate IL-18 secretion from MDDCs but IL-1F8 was not as potent as IL-1F2 in stimulating secretion of IL-12p70 from MDDCs or inducing lymphocyte proliferation Therefore, IL-1Rrp2 expression by some DC subsets may have an important function in the human immune response in vivo via its role in differentiation of inflammatory Th1 lymphocytes.     

Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.     

MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis

The scavenger receptor-A I/II (SR-A) and macrophage receptor with collagenous domain (MARCO) share a common domain organisation and ligand repertoire, including selected polyanions and gram-positive and -negative organisms, but differ in fine specificity of ligand binding, tissue distribution and regulation. Neisseria meningitidis (NM) is a selective ligand for SR-A, but there is evidence for an additional SR-A-independent, polyanion-sensitive component for NM recognition. We therefore studied the relative contribution of MARCO and SR-A to binding of NM by resident and elicited peritoneal macrophages obtained from MARCO-/-, SR-A-/- and SR-A-MARCO-/- mice. Results confirmed that both mouse and human MARCO are able to bind NM independently of NM LPS. MARCO and SR-A contributed independently to NM binding, correlating with their expression levels in different cell populations, but neither of these two molecules was required for release of TNF-alpha and nitric oxide. We propose that the TLR-dependent induction of MARCO by innate immune stimulation enhances recognition and uptake of pathogenic organisms such as NM, thus contributing to host defence against infection.     

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77 %) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.     

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.     

Tissue distribution of the human CD97 EGF-TM7 receptor

CD97 is a founding member of the EGF-TM7 family of class II seven-span transmembrane (7-TM) receptors. CD97 has an extended extracellular region with several N-terminal epidermal growth factor (EGF)-like domains, which mediate binding to CD55. Previous studies demonstrated the expression of CD97 on activated lymphocytes, monocytes, macrophages, granulocytes, and numerous haematopoietic and nonhaematopoietic cell lines. Here, we determined the cellular distribution of human CD97 in situ by immunohistochemistry (IH) and immunofluorescence (IF). Abundant expression of CD97 was detected on all types of macrophages and dendritic cells, except for microglia. Within the lymphoid lineage, most T cells but only a few B cells express CD97. Germinal centre B cells do not express the molecule. Except for smooth muscle cells, no staining was found on other cells outside the immune system. However, analysis of a restricted set of epithelial tumors revealed CD97 expression on the malignant cells in thyroid and gastrointestinal tract cancer.     

F4/80, a monoclonal antibody directed specifically against the mouse macrophage

A hybridoma clone which secretes a macrophage (MΦ)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG_2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.     

Precise mapping of the EGF receptor gene on the human chromosome 7p12 using an improved fish technique

Summary The gene for epidermal growth factor receptor (EGFR) has been localized to the p14-p12 region of human chromosome 7 by somatic cell hybridization and radioisotopein situ hybridization techniques. In this paper, we report the precise mapping of the EGFR gene to the band p12 of chromosome 7 using a novel method in which fluorescence images fromin situ hybridization and Q-banding are computer graphically merged. The novel procedure is described in detail.     

Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX₁ receptors. Altogether ten chimaeras between OX₁ and OX₂ orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K_B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX₂. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX₁ and OX₂ receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.     

HLA-B, -DRB1/3/4/5, and -DQB1 gene polymorphisms in human immunodeficiency virus-related Kaposi's sarcoma

Polymorphisms of genes in the human leukocyte antigen (HLA) complex, particularly those encoding HLA-DR, have been suggested as markers of susceptibility to Kaposi's sarcoma (KS). We conducted a case-control study comparing 147 homosexual men who developed KS after infection by human immunodeficiency virus-1 (HIV-1) and human herpes virus 8 (HHV8) with 147 matched dually infected men without HIV-associated KS (HIV-KS) from the Multicenter AIDS Cohort Study. HLA-B, DRB1, DRB3, DRB4, DRB5, and DQB1 polymorphisms were examined by high-resolution DNA-based methods. Differences in distributions of genetic variants were tested by conditional logistic regression. Previously reported relationships with HLA-DRB1 alleles could not be confirmed. Instead, other associations were observed. In univariate analysis, KS was weakly associated with B*2702/5 (odds ratio (OR)=0.40, 95% confidence interval (CI)=0.18-0.91). Similar or stronger associations, positive or negative, were seen for haplotypes containing class II alleles: DRB1*1302-DQB1*0604 (OR=3.67, 95% CI=1.02-13.1), DRB4 (DR53) haplotype family members [OR=0.52, 95% CI=0.32-0.85], and DRB3 (DR52) haplotype family members (OR=1.69, 95% CI=1.07-2.67). The B*1402-DRB1*0102 haplotype, which invariably contains the V281L mutation in the 21-hydroxylase gene governing adrenal steroid biosynthesis, occurred in five cases and one control (OR=5.0, 95% CI=0.58-42.8). In a final multivariable analysis, only DRB1*1302-DQB1*0604 (OR=6.43, 95% CI=1.28-32.3, P=0.02) remained significantly associated with KS. Associations of HLA-DRB families with HIV-KS could reflect underlying immune dysregulation. The HLA B*1402-DRB1*0102 haplotype associated with increased risk of KS might represent an antigen-presenting pathway unfavorable for immune response to HHV8. Alternatively, the relationship might hold a clue to the predilection of KS for men because that haplotype harbors the mutant form of the 21-hydroxylase gene.     

Molecular cloning of a cDNA encoding the human interleukin 4 receptor

Using the mouse interleukin 4 (IL-4) receptor cDNA as a probe, we isolated a cDNA encoding the human IL-4 receptor (hIL-4 receptor) from a multifactor-responsive human myeloid cell line, TF1. The cDNA encodes for an open reading frame of 825 amino acids including a signal sequence (25 amino acids), the external domain (207 amino acids), a transmembrane domain (24 amino acids), and a large cytoplasmic domain (569 amino acids). The human IL-4 receptor has a 65% identity with the mouse IL-4 receptor at the nucleic acid level and retains the typical structural motif of the previously described cytokine receptor family. COS7 cells transfected with the full-length cDNA expressed high levels (140,000 sites/cell) of IL-4 binding sites, with a Kd = 80 pM, an affinity identical to that of the original TF1 cells. Similar to IL-4 responsive cells, cross-linking of [125I]IL-4 to COS7 cells transfected with the cDNA showed a major protein of 130-150 kd and minor species of 55-85 kd.     

Transient receptor potential vanilloid 1 (TRPV1), TRPV4, and the kidney

Recent preclinical data indicate that activators of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) may improve the outcome of ischaemic acute kidney injury (AKI). The underlying mechanisms are unclear, but may involve TRPV1 channels in dorsal root ganglion neurones that innervate the kidney. Recent data identified TRPV4, together with TRPV1, to serve as major calcium influx channels in endothelial cells. In these cells, gating of individual TRPV4 channels within a four‐channel cluster provides elementary calcium influx (calcium sparklets) to open calcium‐activated potassium channels and promote vasodilation. The TRPV receptors can also form heteromers that exhibit unique conductance and gating properties, further increasing their spatio‐functional diversity. This review summarizes data on electrophysiological properties of TRPV1/4 and their modulation by endogenous channel agonists such as 20‐HETE, phospholipase C and phosphatidylinositide 3‐kinase (PI3 kinase). We review important roles of TRPV1 and TRPV4 in kidney physiology and renal ischaemia reperfusion injury; further studies are warranted to address renoprotective mechanism of vanilloid receptors in ischaemic AKI including the role of the capsaicin receptor TRPV1 in primary sensory nerves and/or endothelium. Particular attention should be paid to understand the kidneys' ability to respond to ischaemic stimuli after catheter‐based renal denervation therapy in man, whereas the discovery of novel pharmacological TRPV modulators may be a successful strategy for better treatment of acute or chronic kidney failure.     

Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(1-7), offers a promising novel treatment modality for this syndrome. As the involvement of ACE and ACE2 in ARDS is still unclear, this study investigated the role of these two enzymes in an animal model of ARDS. ARDS was induced in rats by intratracheal administration of LPS followed by mechanical ventilation. During ventilation, animals were treated with saline (placebo), losartan (Ang II receptor antagonist), or with a protease-resistant, cyclic form of Ang-(1-7) [cAng-(1-7)]. In bronchoalveolar lavage fluid (BALF) of ventilated LPS-exposed animals, ACE activity was enhanced, whereas ACE2 activity was reduced. This was matched by enhanced BALF levels of Ang II and reduced levels of Ang-(1-7). Therapeutic intervention with cAng-(1-7) attenuated the inflammatory mediator response, markedly decreased lung injury scores, and improved lung function, as evidenced by increased oxygenation. These data indicate that ARDS develops, in part, due to reduced pulmonary levels of Ang-(1-7) and that repletion of this peptide halts the development of ARDS.     

B7/BB1, the ligand for CD28, is expressed on repeatedly activated human T cells in vitro

The activation of T cells is now thought to require at least two distinct signals. One signal is delivered through the interaction of the antigen-specific T cell receptor with major histocompatibility complex (MHC) molecules and peptide, while the other is received from interactions with less precisely defined accessory or costimulatory molecules. In the absence of this second costimulatory signal, some T cells subsequently become unresponsive to antigenic stimulation. One of the major candidates for providing such a second signal to T cells is the molecule B7 interacting with the T cell glycoprotein CD28. In the present study we have investigated whether B7 is expressed on human T cell lines and clones, since these cells have the capacity to present antigen to each other by expressing MHC class II molecules. Our results demonstrate that B7 can be detected on T cell clones and on repeatedly activated but not freshly isolated peripheral blood T cells. The expression of B7 is dependent on the state of activation of the cells, being maximally expressed shortly after restimulation and becoming undetectable as the cells quiesce. Together, these results suggest that B7 expression may be of importance to T cells, perhaps in the avoidance of anergy.