David Naor

CD44: Structure, Function and Association with the Malignant Process

CD44 is a ubiquitous multistructural and multifunctional cells surface adhesion molecule involved in cell-cell and cell-matrix interactions. Twenty exons are involved in the genomic organization of this molecule. The first five and the last 5 exons are constant, whereas the 10 exons located between these regions are subjected to alternative splicing, resulting in the generation of a variable region. Differential utilization of the 10 variable region exons, as well as variations in N-glycosylation, O-glycosylation, and glycosaminoglycanation (by heparan sulfate or chondroitin sulfate), generate multiple isoforms (at least 20 are known) of different molecular sizes (85-230 kDa). The smallest CD44 molecule (85-95 kDa), which lacks the entire variable region, is standard CD44 (CD44s). As it is expressed mainly on cells of lymphohematopoietic origin, CD44s is also known as hematopoietic CD44 (CD44H). CD44s is a single-chain molecule composed of a distal extracellular domain (containing, the ligand-binding sites), a membrane-proximal region, a transmembrane-spanning domain, and a cytoplasmic tail. The molecular sequence (with the exception of the membrane-proximal region) displays high interspecies homology. After immunological activation, T lymphocytes and other leukocytes transiently upregulate CD44 isoforms expressing variant exons (designated CD44v). A CD44 isform containing the last 3 exon products of the variable region (CD44V8-10, also known as epithelial CD44 or CD44E), is preferentially expressed on epithelial cells. The longest CD44 isoform expressing in tandem eight exons of the variable region (CD44V3-10) was detected in keratinocytes. Hyaluronic acid (HA), an important component of the extracellular matrix (ECM), is the principal, but by no means the only, ligand of CD44. Other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate. Mucosal addressin, serglycin, osteopontin, and the class II invariant chain (Ii) are additional, ECM-unrelated, ligands of the molecule. In many, but not in all cases, CD44 does not bind HA unless it is stimulated by phorbol esters, activated by agonistic anti-CD44 antibody, or deglycosylated (e.g., by tunicamycin). CD44 is a multifunctional receptor involved in cell-cell and cell-ECM interactions, cell traffic, lymph node homing, presentation of chemokines and growth factors to traveling cells, and transmission of growth signals. CD44 also participates in the uptake and intracellular degradation of HA, as well as in transmission of signals mediating hematopoiesis and apoptosis. Many cancer cell types as well as their metastases express high levels of CD44. Whereas some tumors, such as gliomas, exclusively express standard CD44, other neoplasms, including gastrointestinal cancer, bladder cancer, uterine cervical cancer, breast cancer and non-Hodgkins lymphomas, also express CD44 variants. Hence CD44, particularly its variants, may be used as diagnostic or prognostic markers of at least some human malignant diseases. Furthermore, it has been shown in animal models that injection of reagents interfering with CD44-ligand interaction (e.g., CD44s- or CD44v-specific antibodies) inhibit local tumor growth and metastatic spread. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. It is hoped that identification of CD44 variants expressed on cancer but not on normal cells will lead to the development of anti-CD44 reagents restricted to the neoplastic growth.

CD44 in cancer

CD44 is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines, and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. All these biological properties are essential to the physiological activities of normal cells, but they are also associated with the pathologic activities of cancer cells. Experiments in animals have shown that targeting of CD44 by antibodies, antisense, and CD44-soluble proteins markedly reduces the malignant activities of various neoplasms, stressing the therapeutic potential of anti-CD44 agents. Furthermore, because alternative splicing and posttranslational modifications generate many different CD44 sequences, including, perhaps, tumor-specific sequences, the production of anti-CD44 tumor-specific agents may be a realistic therapeutic approach. However, in many cancers (renal cancer and non-Hodgkins lymphomas are exceptions), a high level of CD44 expression is not always associated with an unfavorable outcome. On the contrary, in some neoplams CD44 upregulation is associated with a favorable outcome. Even worse, in many cases different research groups analyzing the same neoplastic disease reached contradictory conclusions regarding the correlation between CD44 expression and disease prognosis, possibly due to differences in methodology. These problems must be resolved before applying anti-CD44 therapy to human cancers

RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: A different interpretation of redundancy

We report here that joint inflammation in collagen-induced arthritis is more aggravated in CD44-knockout mice than in WT mice, and we provide evidence for molecular redundancy as a causal factor. Furthermore, we show that under the inflammatory cascade, RHAMM (receptor for hyaluronan-mediated motility), a hyaluronan receptor distinct from CD44, compensates for the loss of CD44 in binding hyaluronic acid, supporting cell migration, up-regulating genes involved with inflammation (as assessed by microarrays containing 13,000 cDNA clones), and exacerbating collagen-induced arthritis. Interestingly, we further found that the compensation for loss of the CD44 gene does not occur because of enhanced expression of the redundant gene (RHAMM), but rather because the loss of CD44 allows increased accumulation of the hyaluronic acid substrate, with which both CD44 and RHAMM engage, thus enabling augmented signaling through RHAMM. This model enlightens several aspects of molecular redundancy, which is widely discussed in many scientific circles, but the processes are still ill defined.

Suppressor cells: permitters and promoters of malignancy?

Publisher Summary This chapter discusses the relationships between suppressor cells and tumors. It describes the data that demonstrate the augmentation of antitumor responses in experimental immunocrippled animals (adult thyniectomized, splenectomized, X-irradiated, or antithymocyte serum (ATS)-injected animals). Most of these experiments were performed before the recognition of the function of suppressor cells. The chapter presents the more direct experimental evidence showing the effect of specific and nonspecific suppressor cells on the relationships between the tumor and the host immune system. For the most part, the effects of suppressor cells on syngeneic tumors, and occasionally on the so-called nonspecific tumor cells, are discussed. Nonspecific neoplastic cells cannot stimulate a detectable allogeneic immune response after inoculation into an allogeneic host and they can grow progressively in such an environment. The suppressor cells are classified arbitrarily into permitter suppressor cells—which populate the host before its confrontation with the tumor and promoter suppressor cells—which are induced by the tumor. The chapter also describes the chemical, physical, and biological properties of some of these suppressor cells and offers various means for their selective elimination.

MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.

Immunological and autoimmune considerations of Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) are a group of heterogeneous neurodevelopmental conditions presenting in early childhood with a prevalence ranging from 0.7% to 2.64%. Social interaction and communication skills are impaired and children often present with unusual repetitive behavior. The condition persists for life with major implications for the individual, the family and the entire health care system. While the etiology of ASD remains unknown, various clues suggest a possible association with altered immune responses and ASD. Inflammation in the brain and CNS has been reported by several groups with notable microglia activation and increased cytokine production in postmortem brain specimens of young and old individuals with ASD. Moreover several laboratories have isolated distinctive brain and CNS reactive antibodies from individuals with ASD. Large population based epidemiological studies have established a correlation between ASD and a family history of autoimmune diseases, associations with MHC complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. In addition, there is evidence that antibodies that are only present in some mothers of children with ASD bind to fetal brain proteins and may be a marker or risk factor for ASD. Studies involving the injection of these ASD specific maternal serum antibodies into pregnant mice during gestation, or gestational exposure of Rhesus monkeys to IgG subclass of these antibodies, have consistently elicited behavioral changes in offspring that have relevance to ASD. We will summarize the various types of studies associating ASD with the immune system, critically evaluate the quality of these studies, and attempt to integrate them in a way that clarifies the areas of immune and autoimmune phenomena in ASD research that will be important indicators for future research.

CD44 in rheumatoid arthritis.

CD44 is a multistructural cell-surface glycoprotein that can theoretically generate close to 800 isoforms by differential alternative splicing. At present, several dozen isoforms are known. The polymorphic nature of CD44 might explain its multifunctionality and its ability to interact with many cell-surface and extracellular ligands, the principal one being hyaluronic acid (HA). Of the many CD44 functions, our review focuses on its involvement in cell–cell and cell–matrix interactions, as well as on its implication in the support of cell migration and the presentation of growth factors to their cognate receptors. Cells involved in pathological activities such as cancer cells and destructive inflammatory cells, and also normal cells engaged in physiological functions, use cell-surface CD44 for their localization and expansion at extravascular sites. This article reviews the evidence that the joint synovium of patients with rheumatoid arthritis (RA) contains considerable amounts of various CD44 isoforms as well as the HA ligand. The review also shows that anti-CD44 monoclonal antibody (mAb) directed against constant epitopes, shared by all CD44 isoforms, can markedly reduce the inflammatory activity of arthritis induced by collagen or proteoglycans in mice. Anti-CD44 mAb also interferes with the migration of RA synovial-like fibroblasts in vitro and is able to disturb the destructive interaction between RA synovial-like fibroblasts and the cartilaginous matrix. However, the transition from the experimental model to the patients bedside is dependent on the ability to target the CD44 of cells engaged in RA pathology, while skipping the CD44 of normal cells.

The Involvement of CD44 and Its Novel Ligand Galectin-8 in Apoptotic Regulation of Autoimmune Inflammation

The synovial fluid (SF) cells of rheumatoid arthritis (RA) patients express a specific CD44 variant designated CD44vRA. Using a cellular model of this autoimmune disease, we show in this study that the mammalian lectin, galectin-8 (gal-8), is a novel high-affinity ligand of CD44vRA. By affinity chromatography, flow cytometry, and surface plasmon resonance, we demonstrate that gal-8 interacts with a high affinity (Kd, 6 × 10−9 M) with CD44vRA. We further demonstrate that SF cells from RA patients express and secrete gal-8, to a concentration of 25–65 nM, well within the concentration of gal-8 required to induce apoptosis of SF cells. We further show that not all gal-8 remains freely soluble in the SF and at least part forms triple complexes with CD44 and fibrinogen that can be detected, after fibrinogen immunoprecipitation, with Abs against fibrinogen, gal-8 and CD44. These triple complexes may therefore increase the inflammatory reaction by sequestering the soluble gal-8, thereby reducing its ability to induce apoptosis in the inflammatory cells. Our findings not only shed light on the receptor-ligand relationships between CD44 and gal-8, but also underline the biological significance of these interactions, which may affect the extent of the autoimmune inflammatory response in the SF of RA patients.

CD44 Involvement in Autoimmune Inflammations The Lesson to be Learned from CD44-Targeting by Antibody or from Knockout Mice

Abstract:  CD44 is a multistructural and multifunctional glycoprotein, the diversity of which is generated by alternative splicing. In this communication we review some aspects related to CD44 structure and function in experimental autoimmune inflammation, focusing on research performed in our own laboratory. We have found that CD44 targeting by antibody, passively injected into DBA/1 mice with collagen‐induced arthritis (CIA) and NOD mice with type I diabetes or actively generated by CD44 cDNA vaccination of SJL/j mice with autoimmune encephalomyelitis, markedly reduced the pathological manifestations of these diseases by attenuating cell migration of the inflammatory cells and/or by their apoptotic killing. However, genetic deletion of CD44 by knockout technology enhanced the development of CIA because of molecular redundancy mediated by RHAMM (a receptor of hyaluronan‐mediated motility). The mechanisms that stand behind these findings are discussed.

A mutation in a CD44 variant of inflammatory cells enhances the mitogenic interaction of FGF with its receptor

Synovial fluid cells from joints of rheumatoid arthritis (RA) patients express a novel variant of CD44 (designated CD44vRA), encoding an extra trinucleotide (CAG) transcribed from intronic sequences flanking a variant exon. The CD44vRA mutant was detected in 23 out of 30 RA patients. CD44-negative Namalwa cells transfected with CD44vRA cDNA or with CD44v3-v10 (CD44vRA wild type) cDNA bound FGF-2 to an equal extent via their associated heparan sulfate chains. However, Namalwa cells, immobilizing FGF-2 via their cell surface CD44vRA, bound substantially more soluble FGF receptor-1 (FGFR-1) than did Namalwa cells immobilizing the same amount of FGF-2 via their cell surface CD44v3-v10. The former cells stimulated the proliferation of BaF-32 cells, bearing FGFR-1, more efficiently than did the latter cells. Finally, isolated primary synovial fluid cells from RA patients expressing CD44vRA bound more soluble FGFR-1 to their cell surface-associated FGF-2 than did corresponding synovial cells expressing CD44v3-v10 or synovial cells from osteoarthritis patients. The binding of soluble FGFR-1 to RA synovial cells could be specifically reduced by their preincubation with Abs against the v3 exon product of CD44. Hence, FGF-2 attached to the heparan sulfate moiety expressed by the novel CD44 variant of RA synovium cells exhibits an augmented ability to stimulate FGFR-1-mediated activities. A similar mechanism may foster the destructive inflammatory cascade not only in RA, but also in other autoimmune diseases.