Ligation of human Fc receptor like‐2 by monoclonal antibodies down‐regulates B‐cell receptor‐mediated signalling

B‐cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B‐cell response and function. Human Fc receptor like‐2 (FCRL2), a member of the newly identified FCRL family, could influence B‐cell signalling due to possession of both immunoreceptor tyrosine‐based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2‐specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2‐expressing B‐cell line. Two anti‐FCRL2 mAb‐producing hybridoma clones (5A7‐E7 and 3D8‐G8) were selected. None of the mAbs displayed any cross‐reactivity with the other members of the FCRL family including recombinant FCRL1, ‐3, ‐4 and ‐5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen‐activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti‐FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells.     

β‐Catenin and E‐cadherin expression in stage I adult‐type granulosa cell tumour of the ovary: correlation with tumour morphology and clinical outcome

Aims: To study E‐cadherin and β‐catenin expression in stage I adult‐type granulosa cell tumours (AGCTs) and correlate the findings with tumour morphology and clinical outcome. Methods and results: The study group comprised 62 FIGO stage I AGCTs, including 48 stage IA and 14 stage IC cases. Fifty patients (80.6%) had negative clinical follow‐up over periods from 3.0 to 19.2 years (median 6.4 years), and 12 patients (19.4%) developed metastases at intervals of 3.6–16.2 years (median 8.6 years). β‐Catenin and E‐cadherin were expressed in 62 (100%) and 53 (85%) primary tumours, respectively, and staining was more consistent and intense in areas showing sex cord‐like morphology. In contrast, diffuse tumour areas often showed weak or moderate staining (β‐catenin) or were negative (E‐cadherin), and there was reduced expression of both proteins in luteinized cells. Reduced β‐catenin expression in primary tumours correlated with increased risk of recurrence (P = 0.002) and a shorter time interval to recurrence, whereas there was no correlation between E‐cadherin staining and the risk of metastases. Conclusions: Localized variations in adhesion protein expression may partly explain the diverse morphological patterns exhibited by AGCT, and reduced β‐catenin staining in primary tumours may have value as an adverse prognostic factor.     

Dose‐dependent effects of prostaglandin E2 in macrophage adhesion and migration

Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE₂), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose‐dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE₂ increased p110γ PI3K expression, phosphorylation of actin‐related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE₂ increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE₂ as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE₂.     

Critical role of the NKG2D receptor for NK cell‐mediated control and immune escape of B‐cell lymphoma

Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ‐myc‐transgenic model of endogenously arising lymphoma and NKG2D‐deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN‐γ expression, which was effected by MHCI^low tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D‐deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D‐deficient λ‐myc‐transgenic mice was significantly accelerated compared to NKG2D‐competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D‐L) in late disease stages, this did not occur in NKG2D‐deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D‐L loss mutants provides a mechanism of tumor escape.     

LRIG1 regulates cadherin‐dependent contact inhibition directing epithelial homeostasis and pre‐invasive squamous cell carcinoma development

Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine‐rich immunoglobulin repeats‐1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re‐expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E‐cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition. © 2012 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Sympathetic neural signaling via the β2‐adrenergic receptor suppresses T‐cell receptor‐mediated human and mouse CD8+ T‐cell effector function

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2‐adrenergic receptor (ADRB2) on primary human CD8⁺ effector memory T cells. Treatment of both human and murine CD8⁺ T cells with NE decreased IFN‐γ and TNF‐α secretion and suppressed their cytolytic capacity in response to T‐cell receptor (TCR) activation. The effects of NE were specifically reversed by β 2‐specific antagonists. Adrb2^?/? CD8⁺ T cells were completely resistant to the effects of NE. Further, the ADRB2‐specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8⁺ T cells. While both TCR activation and stimulation with IL‐12 + IL‐18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN‐γ secretion in response to IL‐12 + IL18. Finally, the long‐acting ADRB2‐specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8⁺ T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8⁺ T‐cell function and underscores the novel role this pathway plays in adaptive T‐cell responses to infection.     

CEACAM1 on activated NK cells inhibits NKG2D‐mediated cytolytic function and signaling

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1‐bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin‐2‐induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1‐bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans‐homophilic interactions between CEACAM1. Mechanistically, co‐engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D‐mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.     

Polymorphonuclear neutrophils promote dyshesion of tumor cells and elastase‐mediated degradation of E‐cadherin in pancreatic tumors

Pancreatic ductal adenocarcinoma (PDAC) presenting with a micropapillary growth pattern is frequently associated with a prominent neutrophil infiltration into the tumor. The relevance of neutrophil infiltrates for tumor progression, however, is still debated. To gain insight into the role of polymorphonuclear neutrophils (PMNs) in PDAC, we assessed their effect on pancreatic tumor cells grown in vitro as monolayers. Time‐lapse video microscopy showed a PMN‐induced dyshesion of the tumor cells, and subsequent experiments revealed that this dyshesion was due to PMN elastase‐mediated degradation of E‐cadherin, an adhesion molecule that mediates the intercellular contact of the tumor cells. E‐cadherin degradation by elastase or — (for comparison) down‐modulation by specific siRNA, significantly increased the migratory capacity of the pancreatic tumor cells, leading to the hypothesis that PMNs could contribute to the invasive tumor growth. To address this issue, biopsies of patients with PDAC (n = 112) were analyzed. We found that E‐cadherin expression correlated negatively with PMN infiltration, compatible with the notion that E‐cadherin is cleaved by PMN‐derived elastase, which in turn could result in the dispersal of the tumor cells, enhanced migratory capacity and thus invasive tumor growth.     

Immunohistochemical analysis of the expression of E‐cadherin and ZEB1 in non‐small cell lung cancer

We performed an immunohistochemical analysis of the expression of zinc‐finger E‐box binding homeobox 1 (ZEB1), a master regulator of epithelial‐mesenchymal transition (EMT), and determined its relationship with E‐cadherin in 157 non‐small cell lung carcinomas (93 adenocarcinomas, 36 squamous cell carcinomas, 18 large cell carcinomas, and 10 pleomorphic carcinomas). Although the expression of E‐cadherin was low in the subset of adenocarcinomas (10%) and squamous cell carcinomas (11%), ZEB1 expression was only observed in one case of squamous cell carcinoma and none of the adenocarcinomas. In contrast, the low expression of E‐cadherin (50% and 90%, respectively) and the positive expression of ZEB1 (11% and 50%, respectively) were more frequently observed in poorly differentiated carcinomas (large cell carcinomas and pleomorphic carcinomas). Overall, the expression of ZEB1 was inversely correlated with that of E‐cadherin. Furthermore, the distribution of ZEB1‐positive cancer cells was more restricted than in the area in which the expression of E‐cadherin was lost, and the former was detected within the latter. We concluded that the expression of ZEB1 was not necessarily associated with the low expression of E‐cadherin in lung adenocarcinomas and squamous cell carcinomas. The expression of ZEB1 correlated with an undifferentiated and/or sarcomatoid morphology that may occur in the late stage of EMT.     

Stromal CD10 expression and relationship to the E‐cadherin/β‐catenin complex in breast carcinoma

Kim H‐S, Kim G Y, Kim Y W, Park Y‐K, Song J‐Y & Lim S‐J
(2010) Histopathology 56, 708–719
Stromal CD10 expression and relationship to the E‐cadherin/β‐catenin complex in breast carcinoma Aims: Previous investigations have indicated that stromal CD10 expression, and altered levels of both E‐cadherin and β‐catenin, are associated with the biological aggressiveness of human carcinoma. The aim was to evaluate stromal CD10 expression and the association of stromal CD10 with E‐cadherin and β‐catenin in breast carcinoma. Methods and results: The expression of CD10, E‐cadherin and β‐catenin was immunohistochemically analysed in tissue microarrays containing 104 cases of invasive ductal carcinoma (IDC) and 10 cases of ductal carcinoma in situ (DCIS). Stromal CD10 was detected in 49.5% (50/101) of the IDC. No immunoreactivity was identified in the stromal cells of normal breast, DCIS or intraductal components of IDC. Accumulation of the cytoplasmic β‐catenin was found in 87.0% (87/100) of the IDC. Stromal CD10 expression in IDC was significantly correlated with tumour size (P = 0.027), stage (P < 0.001) and histological grade (P = 0.006), the presence of nodal (P = 0.048) and distant (P = 0.015) metastases, oestrogen receptor‐negative status (P = 0.016), cytoplasmic β‐catenin accumulation (P = 0.031) and lower overall survival rate (P = 0.041). Conclusions: Stromal CD10 expression in IDC may constitute an important prognostic marker. Stromal CD10 expression with associated aggressive features might be related to aberrant β‐catenin expression.     

Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2‐specific D32.10 monoclonal antibody

The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297–306, E2:480–494, and E2:613–621) expressed on the surface of serum‐derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell‐binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand–receptor binding data. HCV adsorption was also assessed by quantitating cell‐associated viral RNA by a real‐time RT‐PCR method. Saturable concentration‐dependent specific binding of HCVsp to Huh‐7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two‐site interaction for Huh‐7 and proliferative HepaRG cells: the high‐affinity binding sites (K_d1 = 0.1–0.5 µg/ml) and the low‐affinity binding sites (K_d1 = 5–10 µg/ml), and one‐site high‐affinity binding model between E1E2/D32.10‐positive HCVsp and hepatocyte‐like differentiated HepaRG cells. The E1E2‐specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC₅₀ ≤0.5 µg/ml in all the experimental approaches using serum HCV of genotype either 3 or 1b. This supports the involvement of the E1E2/D32.10 discontinuous antigenic determinant in the interactions between human hepatocytes and HCVsp, and suggests that D32.10‐like antibodies present in sera from patients infected with HCV could play a protective role. J. Med. Virol. 81:1726–1733, 2009. © 2009 Wiley‐Liss, Inc.     

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y₇F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1⁺ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.