Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling.

Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose-capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-gamma) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production.

ALK-positive anaplastic large-cell lymphoma: strong T and B anti-tumour responses may cause hypocellular aspects of lymph nodes mimicking inflammatory lesions.

The anaplastic large cell lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a rare type of non-Hodgkin lymphoma which occurs in children mostly. The ALK protein is highly immunogenic and elicits both humoral and cellular immune responses. A 15-yr-old child presented with fever and adenopathy and did not respond to antibiotics. Biopsy of the enlarged lymph node contained almost no lymphoid element except for a few CD8-positive T cells, plasma cells and isolated CD30-positive blasts. The patient's condition improved following lymphadenectomy but relapse occurred 3 months later with multiple nodes, high fever and an abdominal mass. This time an ALK-positive ALCL was diagnosed and the retrospective analysis of the initial biopsy revealed rare, isolated ALK+ cells. Molecular analysis showed T-cell clones and oligoclonal B cells in both biopsies and peripheral blood of the patient. The tumour cells harbour a t(2;5) translocation, revealing a null phenotype by immunohistochemistry and no evidence for T-cell clonality by Southern blotting. The patient's serum contained anti-ALK antibodies. Our findings suggest that the T-cell clones and anti-ALK antibodies in this patient constitute an anti-tumour response that caused the hypocellularity of the initial lymph node. Hypocellular and oedematous lymph nodes occurring in a child with evocative symptoms should be tested for the presence of ALK.

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.

Anti-CD20 treatments and the lymphocyte membrane: pathology for therapy.

Therapeutic antibodies directed against the CD20 surface protein of B-lymphocytes are efficient means of controlling the growth and survival of malignant B-lymphocytes. The mechanisms of anti-CD20 antibody action remain in great part unexplained. However, we show that incubation of CD20+ cells with therapeutic antibodies such as Rituximab results in the redistribution of the CD20 marker in plasma membrane rafts. Rafts are specialized membrane organizations of sphingolipids and cholesterol in the plasma membrane outer leaflet that serve as signalling platforms in lymphocytes and other cells, and allow transmembrane propagation of most receptor-mediated extracellular signals. This redistribution of CD20 to rafts is not acutely toxic to lymphoma cells, but leads to long-lasting perturbations of transmembrane signalling and contributes to the progressive elimination of B-lymphoma cells. The accumulation of CD20 in rafts causes downmodulation of raft-associated protein tyrosine kinases and modifies the spatial relationships between raft lipid and protein components. Such modifications of the raft structure and function are likely to cause relatively long-lasting perturbations of the lymphoma cell physiology and contribute to the elimination of the Rituximab-targeted cells.

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform.

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners and temporary elaboration of supramolecular structures, to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular nonreceptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor. The concept of the signaling platform has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling. The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.

Signal transduction via CD44: role of plasma membrane microdomains.

CD44 is the principal cell surface receptor for extracellular matrix glycosaminoglycan hyaluronan. CD44-hyaluronan mediated cell adhesion is important in several pathophysiological processes such as inflammation and metastatic spread of cancer cells. It has been recently recognized that CD44 also functions as a signaling receptor in a variety of cell types. Cell stimulation by monoclonal anti-CD44 antibody or natural CD44 ligands activate several signaling pathways that culminate in cell proliferation, cytokine secretion, chemokine gene expression and cytolytic effector functions. One of the earliest signaling events following stimulation via CD44 is tyrosine phosphorylation of intracellular proteins substrates, and CD44 mediated cellular activation could be abolished by protein tyrosine kinase (PTK) inhibitors. The Src-family non-receptor PTKs such as Lck, Fyn, Lyn and Hck were shown to be coupled to CD44 via sphingolipid-rich microdomains (lipid rafts) of the plasma membrane. Studies on T cell receptor and IgE receptor mediated signaling in lymphocytes and mast cells have consolidated the notion that microdomains consist of signaling platforms where components of multiple signaling pathways are assembled. Co-isolation of CD44 with microdomains strongly suggests that CD44 generates cellular activation signals utilizing the signaling machinery of the plasma membrane microdomains.

Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes.

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.

Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.

CD44 selectively associates with active Src family protein tyrosine kinases Lck and Fyn in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes.

CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

Carbohydrate moiety of Plasmodium falciparum glycoproteins: the nature of the carbohydrate-peptide linkage in the MSP-2 glycoprotein.

Metabolic labelling of Plasmodium falciparum parasites with [3H]GlcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS-PAGE of the labelled material provided effective labelling of the MSP-1, 195 kDa, and MSP-2, 42-53 kDa, glycoproteins. Reductive beta-elimination of the MSP-2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N-acetylglucosaminitol and N-acetylgalactosaminitol amongst other labelled sugars. Reductive beta-elimination with sodium hydroxide-sodium borotritide-borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP-2 was retained on solid phase wheat-germ agglutinin and was released from the lectin by treatment with GlcNAc. Upon treatment with O-glycanase the MSP-2 glycoprotein released labelled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP-2 glycoprotein using [3H]UDP-Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with beta-galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP-2 glycoprotein are attached to the protein backbone via GlcNAc- and GalNAc-serine/threonine in O-glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP-2, glycoprotein consist mainly of short chains linked to the protein core.

Distinct interactions among GPI-anchored, transmembrane and membrane associated intracellular proteins, and sphingolipids in lymphocyte and endothelial cell plasma membranes.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins are enriched in sphingolipid-rich plasma membrane domains, which are often isolated as low-density membrane complexes. This association is believed to arise from the interactions between the GPI-acyl chains and sphingolipids, but is not fully understood. In this study, we compared the physical properties of GPI-anchored glycoproteins from a non-polarized (murine T-lymphocyte) and a polarized (human endothelial) cell by equilibrium density gradient centrifugation after extraction by detergents under identical conditions. Unlike those on epithelial cells, the GPI-anchored proteins of lymphocytes (Thy-1 and the heat stable antigen CD24) were enriched in the floating fractions after extraction over a wide range of octylglucoside concentrations. In contrast, the floatability of endothelial GPI-anchored CD59 was markedly diminished, not only by octylglucoside, but also by increasing concentrations of Triton X-100. Distribution of cholera toxin binding ganglioside GM1 in the sucrose gradient fractions closely followed that of the GPI-anchored proteins in both lymphocytes and endothelial cells under most extraction conditions. Analysis of the intracellular acylated molecules revealed that a significant amount of p56(lck) was always associated with the floating GPI-rich fractions of lymphocytes when extracted by Triton X-100 or octylglucoside at 4 degrees C, while the behaviour of endothelial cell caveolin was comparable to that of CD59. The transmembrane glycoproteins CD45 in lymphocytes and MHC class I antigen in endothelial cells interacted weakly with GPI domains, whereas endothelial CD44 and lymphocyte CD26 displayed a strong association. These results show that: (1) the physical properties of different GPI-anchored proteins may vary significantly; and (2) transmembrane and acylated intracellular proteins could be associated with GPI domains to a variable extent. These differences probably reflect cell type-specific interactions of GPI anchors with the sphingolipid framework of plasma membranes, as well as extracellular interactions of GPI-anchored glycoproteins with neighbouring cell surface molecules.

Differential regulation of Src-family protein tyrosine kinases in GPI domains of T lymphocyte plasma membranes.

The association of glycosylphosphatidylinositol (GPI)-anchored cell surface glycoproteins with Src-family protein tyrosine kinases was analysed in intact T lymphocyte plasma membranes. Following subcellular fractionation without detergent, 25% of the recovered plasma membranes were light density vesicles enriched in GPI-anchored glycoproteins and sphingolipids (GPI domains), while the remainder behaved as heavier density vesicles containing equal amounts of lipids and proteins. Qualitatively similar lipids were found in both vesicle types, but only light density vesicles made of 65-75% lipids yielded a Triton X-100 resistant, sedimentable fraction containing GPI-linked glycoproteins and sphingolipids. The GPI-rich vesicles phosphotyrosylated an exogenous substrate as efficiently as the denser vesicles, despite a low Lck and Fyn kinase content. Likewise, these kinases were more efficiently phosphorylated in GPI domains than in denser vesicles. GPI domains thus could constitute plasma membrane "hot spots" where associated Src kinases assume an optimally active conformation that contributes to signaling via GPI-anchored cell surface glycoproteins.

Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen.

Field medicine in tropical areas needs laboratory assays which are inexpensive and easy to perform. To meet this need a semi-quantitative dot-enzyme immunoassay (EIA) was developed for the detection of an L. donovani-related circulating antigen and tested for clinical relevance in the diagnosis of visceral leishmaniasis (VL). The dot-EIA probes serum spotted on nitrocellulose for the presence of the antigen using a monoclonal antibody raised against L. donovani promastigotes, a peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum and chloronaphthol as peroxidase substrate. The intensity of dot staining by chloronaphthol is read by eye and scored. The dot-EIA was used to test the following groups: 69 patients with VL from Brazil, Kenya, China and France, nine patients with cutaneous leishmaniasis, 38 patients with tropical diseases other than VL, five patients with rheumatoid arthritis and 40 health blood donors. The specificity of the assay was 96.7% (3/92 false positive) and the sensitivity 98.5% (1/69 false negative). A quantitative EIA for the detection of serum antibodies which makes use of a crude, soluble L. infantum promastigote extract as capture antigen and which was used as the reference method, proved to be more specific (98.9%) but similarly sensitive (98.5%). It should be possible to produce a kit, suitable for large scale application at low cost in order to facilitate routine use of the dot-EIA in the diagnosis of VL.

Transfer of exogenous glycosylphos-phatidylinositol (GPI)-linked molecules to plasma membranes.

Purified GPI-linked molecules incorporate spontaneously in vitro into mammalian cell plasma membranes. Recent evidence suggests that the transferred molecules insert stably into the external leaflet of the acceptor cell plasma membrane through their acyl chains and behave subsequently in a way similar to endogenous GPI-linked molecules. Transfer of GPI-linked proteins between cells has also been documented in vivo and may explain the uptake by host cells o f pathogen-derived virulence factors carrying a GPI anchor. In this comment article, Subburaj Ilangumaran, Peter Robinson and Daniel Hoessli review what is known about GPI transfer and discuss the use of GPI transfer for transient cell-surface expression of foreign proteins.

Requirement of Lyn and Syk tyrosine kinases for the prevention of apoptosis by cytokines in human eosinophils.

In allergic diseases, the cytokines interleukin (IL)5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are upregulated and have been proposed to cause blood and tissue eosinophilia by inhibition of eosinophil apoptosis. We demonstrate herein, in freshly isolated human eosinophils, that the IL-3/IL-5/GM-CSF receptor beta subunit interacts with cytoplasmic tyrosine kinases to induce phosphorylation of several cellular substrates, including the beta subunit itself. The Lyn and Syk intracellular tyrosine kinases constitutively associate at a low level with the IL-3/IL-5/GM-CSF receptor beta subunit in human eosinophils. Stimulation with GM-CSF or IL-5 results in a rapid and transient increase in the amount of Lyn and Syk associated with the IL-3/IL-5/GM-CSF receptor beta subunit. Lyn is required for optimal tyrosine phosphorylation and activation of Syk. In contrast, Syk is not required for optimal tyrosine phosphorylation and activation of Lyn. These data suggest that Lyn is proximal to Syk in a tyrosine kinase cascade that transduces IL-3, IL-5, or GM-CSF signals. Compatible with this model, both Lyn and Syk are essential for the activation of the antiapoptotic pathway(s) induced through the IL-3/IL-5/GM-CSF receptor beta subunit in human eosinophils.

Evaluation by dot-immunoassay of the differential distribution of cell surface and intracellular proteins in glycosylphosphatidylinositol-rich plasma membrane domains.

The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into specialized plasma membrane domains enriched in glycosphingolipids. The dot-immunoassay has been successfully used to demonstrate the differential distribution of GPI-linked and transmembrane surface glycoproteins of T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein CD26 and the intracellular tyrosine kinase p56lck partially cofractionated with GPI-linked glycoproteins, and the extent to which they partition into GPI-rich plasma membrane domains could be evaluated. Preferential association of the acidic glycosphingolipid GM1 with these domains could be demonstrated by cholera toxin binding directly to the dot-blotted sucrose density gradient fractions. Treatment of whole cell TX-100 lysates or sucrose gradient fractions dotted onto nitrocellulose filter strips with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) proved to be an efficient method to assay for the presence of a GPI-anchor in Thy-1 and Ly6 surface glycoproteins. We have used three criteria, namely flotation to light density fractions in sucrose gradients, colocalization with GM1, and sensitivity to PI-PLC cleavage, to assess the presence of a GPI modification in a putative GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the contents of GPI-rich plasma membrane domains in different cells and tissues.

Integration of mycobacterial lipoarabinomannans into glycosylphosphatidylinositol-rich domains of lymphomonocytic cell plasma membranes.

Lipoarabinomannans (LAMs) are major Ags of the mycobacterial cell envelope where they apparently insert through a glycosylphosphatidylinositol (GPI) anchoring structure. LAMs induce host macrophages to secrete TNF-alpha, IL-1, and IL-6 and inhibit T cell proliferative responses. The mechanisms by which LAMs mediate these effects remain poorly understood. We show that LAMs were efficiently inserted into the plasma membranes of human and murine lymphomonocytic cells through their GPI anchor. Prior deacylation of LAMs abrogated this event. Phosphatidylinositol hexamannoside (PIM6), the GPI anchor of all LAMs, competitively inhibited LAM insertion. Deacylated PIM6 was not inhibitory. The hexamannoside glycan of PIM6 appears to be important for LAM insertion, since phosphatidylinositol from soybean, lacking the glycan core, was not as efficient an inhibitor. Interaction of LAM with target cells was influenced by the gel/fluid phase distribution of membrane lipids, suggesting a direct interaction of the LAM-GPI anchor with the membrane bilayer. The inserted LAMs were mobile in the plane of the membrane and interfered with Ab-mediated mobilization of the GPI-anchored Thy-1 molecules. Further, LAMs were preferentially incorporated into isolated plasma membrane vesicles enriched in Thy-1. Our results strongly suggest that 1) interaction of LAMs with host lymphomonocytic cells is mediated through a preferential integration of LAM-GPI anchor into specialized plasma membrane domains enriched in endogenous GPI-anchored molecules, and 2) both the acyl chains and the mannoside core glycan of the LAM-GPI anchor contribute to the specificity of integration.