A Lymphotoxin/Type I IFN Axis Programs CD8+ T Cells To Infiltrate a Self-Tissue and Propagate Immunopathology.

Type I IFNs (IFN-I) are cytokines that can mediate both immune suppression and activation. Dendritic cells (DC) are significant producers of IFN-I, and depending on the context (nature of Ag, duration of exposure to Ag), DC-derived IFN-I can have varying effects on CD8(+) T cell responses. In this study, we report that in the context of a CD8(+) T cell response to a self-Ag, DC-intrinsic expression of IFN regulatory factor 3 is required to induce optimal proliferation and migration of autoreactive CD8(+) T cells, ultimately determining their ability to infiltrate a target tissue (pancreas), and the development of glucose intolerance in rat insulin promoter-glycoprotein (RIP-GP) mice. Moreover, we show that signals through the lymphotoxin-ß receptor (LTßR) in DC are also required for the proliferation of autoreactive CD8(+) T cells, the upregulation of VLA4/LFA1 on activated CD8(+) T cells, and their subsequent infiltration into the pancreas both in vitro and in vivo. Importantly, the defects in autoreactive CD8(+) T cell proliferation, accumulation of CD8(+) T cells in the pancreas, and consequent glucose intolerance observed in the context of priming by LTßR(-/-) DC could be rescued by exogenous addition of IFN-I. Collectively, our data demonstrate that the LTßR/IFN-I axis is essential for programming of CD8(+) T cells to mediate immunopathology in a self-tissue. A further understanding of the IFN-I/LTßR axis will provide valuable therapeutic insights for treatment of CD8(+) T cell-mediated autoimmune diseases.

Distinct binding affinities of Mac-1 and LFA-1 in neutrophil activation.

Macrophage-1 Ag (Mac-1) and lymphocyte function-associated Ag-1 (LFA-1), two ß2 integrins expressed on neutrophils (PMNs), mediate PMN recruitment cascade by binding to intercellular adhesive molecule 1. Distinct functions of LFA-1-initiating PMN slow rolling and firm adhesion but Mac-1-mediating cell crawling are assumed to be governed by the differences in their binding affinities and kinetic rates. In this study, we applied an adhesion frequency approach to compare their kinetics in the quiescent and activated states using three molecular systems, constitutively expressed receptors on PMNs, wild-type and high-affinity (HA) full-length constructs transfected on 293T cells, and wild-type and HA recombinant extracellular constructs. Data indicate that the difference in binding affinity between Mac-1 and LFA-1 is on-rate dominated with slightly or moderately varied off-rate. This finding was further confirmed when both ß2 integrins were activated by chemokines (fMLF or IL-8), divalent cations (Mg(2+) or Mn(2+)), or disulfide bond lockage on an HA state. Structural analyses reveal that such the kinetics difference is likely attributed to the distinct conformations at the interface of Mac-1 or LFA-1 and intercellular adhesive molecule 1. This work furthers the understandings in the kinetic differences between Mac-1 and LFA-1 and in their biological correlations with molecular activation and structural bases.

Conjugated linoleic acid targets ß2 integrin expression to suppress monocyte adhesion.

Chronic recruitment of monocytes and their subsequent migration through the activated endothelium contribute to atherosclerotic plaque development. Integrin-mediated leukocyte adhesion is central to this process. Conjugated linoleic acid (CLA) has the unique property of inducing regression of pre-established murine atherosclerosis via modulation of monocyte/macrophage function. Understanding the mechanisms through which CLA mediates its atheroprotective effect may help to identify novel pathways that limit or reverse atherosclerosis. In this study, we identified a novel mechanism through which CLA alters monocyte function. We show that CLA inhibits human peripheral blood monocyte cell adhesion to activated endothelial cells via loss of CD18 expression, the ß2 chain of LFA-1 and Mac-1 integrins. In addition, using a static-adhesion assay, we provide evidence that CLA prevents monocytes from binding to ICAM-1 and subsequently reduces the capacity of these cells to polarize. CXCL12-CXCR4 interactions induce a conformational change in ß2 integrins, facilitating leukocyte adhesion. In this study, we demonstrate that CLA inhibits CXCR4 expression, resulting in a failure of monocytes to directionally migrate toward CXCL12. Finally, using intravital microscopy, we show that, during CLA-induced regression of pre-established atherosclerosis in ApoE(-/-) mice, there is reduced leukocyte adhesion and decreased CD18 expression on Gr1(+)/CD115(+) proinflammatory monocytes. In summary, the data presented describe a novel functional role for CLA in the regulation of monocyte adhesion, polarization, and migration.

The tumor promoter and NF-κB modulator Bcl-3 regulates splenic B cell development.

Bcl-3 is an atypical member of the family of IκB proteins. Unlike the classic members, Bcl-3 functions as a nuclear transcriptional cofactor that may, depending on context, promote or suppress genes via association with p50/NF-κB1 or p52/NF-κB2 homodimers. Bcl-3 is also an oncogene, because it is a partner in recurrent translocations in B cell tumors, resulting in deregulated expression. Bcl-3 functions, however, remain poorly understood. We have investigated the role of Bcl-3 in B cells and discovered a previously unknown involvement in the splenic development of these cells. Loss of Bcl-3 in B cells resulted in significantly more marginal zone (MZ) and fewer follicular (FO) B cells. Conversely, transgenic expression of Bcl-3 in B cells generated fewer MZ and more FO B cells. Both Bcl-3(-/-) FO and MZ B cells were more responsive to LPS stimulation compared with their wild-type counterparts, including increased proliferation. By contrast, Bcl-3(-/-) FO B cells were more prone to apoptosis upon BCR stimulation, also limiting their expansion. The data reveal Bcl-3 as a regulator of B cell fate determination, restricting the MZ path and favoring the FO pathway, at least in part, via increased signal-specific survival of the latter, a finding of relevance to its tumorigenic activity.

Coxsackievirus B3 regulates T-cell infiltration into the heart by lymphocyte function-associated antigen-1 activation via the cAMP/Rap1 axis.

Coxsackievirus B3 (CVB3) infection can trigger myocarditis and can ultimately lead to dilated cardiomyopathy. It is known that CVB3-induced T-cell infiltration into cardiac tissues is one of the pathological factors causing cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T-cell infiltration by two types of CVB3: the H3 WT strain and the YYFF attenuated strain. T-cell activation was confirmed by changes in the distribution of lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 could infect and activate T-cells in vivo and in vitro, and activated T-cells were detected in CVB3-infected mouse hearts. LFA-1 expressed on the surface of these T-cells had been activated through the cAMP/Rap1 pathway. Recombinant lentiviruses expressing VP2 of CVB3 could also induce LFA-1 activation via an increase in cAMP, whilst VP2 of YYFF did not. These results indicated that CVB3 infection increased cAMP levels and then activated Rap1 in T-cells. In particular, VP2, among the CVB3 proteins, might be critical for this activation. This VP2-cAMP-Rap1-LFA-1 axis could be a potential therapeutic target for treating CVB3-induced myocarditis.

CD28 controls the development of innate-like CD8+ T cells by promoting the functional maturation of NKT cells.

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αß NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger(+) IL-4(+) NKT cells and upregulation of LFA-1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28-deficient mice is cell autonomous. Moreover, we show in both wild-type C57BL/6 mice and in downstream of tyrosine kinase-1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28-mediated regulation of thymic IL-4(+) NKT cells promotes the differentiation of eomesodermin(+) CD44(high) innate-like CD8(+) T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT-cell homeostasis and the size of the innate-like CD8(+) T-cell pool.

Identification of endothelial cell junctional proteins and lymphocyte receptors involved in transendothelial migration of human effector memory CD4+ T cells.

Human effector memory (EM) CD4(+) T cells can rapidly transmigrate across an endothelial cell (EC) monolayer in response either to chemokine or to TCR-activating signals displayed by human dermal microvascular EC under conditions of venular shear stress. We previously reported that the TCR-stimulated transendothelial migration (TEM) depends on fractalkine (CX3CL1), PECAM-1 (CD31), and ICAM-1 (CD54) expression by the EC, whereas chemokine-stimulated TEM does not. In this study, we further analyze these responses using blocking mAb and small interfering RNA knockdown to show that TCR-stimulated TEM depends on CD99 on EC as well as on PECAM-1 and depends on nectin-2 (CD112) and poliovirus receptor (CD155) as well as EC ICAM-1. ICAM-1 is engaged by EM CD4(+) T cell LFA-1 (CD11a/CD18) but not Mac-1 (CD11b/CD18); nectin-2 and poliovirus receptor are engaged by both DNAX accessory molecule-1 (CD226) and Tactile (CD96). EC junctional adhesion molecule-1 (JAM-1), an alternative ligand for LFA-1, contributes exclusively to chemokine-stimulated TEM and ICAM-2 appears to be uninvolved in either pathway. These data further define and further highlight the differences in the two pathways of EM CD4(+) T cell recruitment into sites of peripheral inflammation.

SKAP1 protein PH domain determines RapL membrane localization and Rap1 protein complex formation for T cell receptor (TCR) activation of LFA-1.

Although essential for T cell function, the identity of the T cell receptor (TCR) "inside-out" pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.

Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes.

During the course of homing to lymph nodes (LNs), T cells undergo a multistep adhesion cascade that culminates in a lymphocyte function-associated antigen 1 (LFA-1)-dependent firm adhesion to the luminal surface of high endothelial venules (HEVs). The importance of LFA-1 affinity regulation in supporting T-cell arrest on HEVs has been well established, however, its importance in the postadhesion phase, which involves intraluminal crawling and diapedesis to the extravascular space, remains elusive. Here we have shown that LFA-1 affinity needs to be appropriately regulated to support these essential steps in the homing cascade. Genetically engineered T cells that were unable to properly down-regulate LFA-1 affinity underwent enhanced, chemokine-independent arrest in HEVs but showed perturbed intravascular crawling to transmigration sites and compromised diapedesis across HEVs. By contrast, the extravascular migration of T cells was insensitive to the affinity-enhancing LFA-1 mutation. These results highlight the requirement for balanced LFA-1 affinity regulation in intravascular and transvascular, but not extravascular, T-cell migration in LNs.

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro.

BACKGROUND: Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. METHODS: A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. RESULTS: We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. CONCLUSION: Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ.

To exit blood vessels, most (∼80%) of the lumenally adhered monocytes and neutrophils crawl toward locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle, we separately examined the crawling and emigration patterns of monocytes and neutrophils in blood-perfused unstimulated or TNF-α-activated venules. Most of the interacting cells in microvessels are neutrophils; however, in unstimulated venules, a greater percentage of the total monocyte population is adherent compared with neutrophils (58.2 ± 6.1% versus 13.6 ± 0.9%, adhered/total interacting), and they crawl for significantly longer distances (147.3 ± 13.4 versus 61.8 ± 5.4 µm). Intriguingly, after TNF-α activation, monocytes crawled for significantly shorter distances (67.4 ± 9.6 µm), resembling neutrophil crawling. Using function-blocking Abs, we show that these different crawling patterns were due to CD11a/CD18 (LFA-1)- versus CD11b/CD18 (Mac-1)-mediated crawling. Blockade of either Mac-1 or LFA-1 revealed that both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1-dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation, likely due to increased expression of Mac-1. Mac-1 alone was responsible for neutrophil crawling in both unstimulated and TNF-α-activated venules. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared with LFA-1) was also significantly more efficient in blocking TNF-α-induced extravasation of both monocytes and neutrophils in cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of leukocytes that transmigrate.

Neutrophils in a mouse model of autoantibody-mediated arthritis: critical producers of Fc receptor gamma, the receptor for C5a, and lymphocyte function-associated antigen 1.

OBJECTIVE: Neutrophils represent a prominent component of inflammatory joint effusions and are required for synovial inflammation in mouse models, but the mechanisms are poorly understood. In this study, we developed a system with which to test the importance of the production of specific factors by neutrophils in a mouse model of arthritis. METHODS: Neutrophil-deficient Gfi-1(-/-) mice were administered sublethal doses of radiation and were then engrafted with donor bone marrow cells (BMCs), which resulted in the production of mature neutrophils within 2 weeks. By reconstituting with BMCs from mice lacking selected proinflammatory factors, we generated mice that specifically lacked these factors on their neutrophils. Arthritis was initiated by transfer of K/BxN serum to identify the role of defined neutrophil factors on the incidence and severity of arthritis. RESULTS: Neutrophils lacking the signaling chain of stimulatory Fc receptors (FcRgamma(-/-)) were unable to elicit arthritis, but neutrophils lacking FcgammaRIII still did so. Neutrophils lacking the chemotactic or adhesion receptor C5a receptor (C5aR) or CD11a/lymphocyte function-associated antigen 1 (LFA-1) also failed to initiate arthritis but could enter joints in which inflammation had been initiated by wild-type neutrophils. Neutrophils unable to produce interleukin-1alpha (IL-1alpha) and IL-1beta (IL-1alpha/beta(-/-)) or leukotrienes (5-lipoxygenase [5-LOX(-/-)]) produced arthritis of intermediate severity. The inability of neutrophils to make tumor necrosis factor or to express receptors for tumor necrosis factor or IL-1 had no effect on arthritis. CONCLUSION: A novel transfer system was developed to identify neutrophil production of FcRgamma, C5aR, and CD11a/LFA-1 as critical components of autoantibody-mediated arthritis. Neutrophil production of IL-1 and leukotriene B(4) likely contributes to inflammation but is not essential. Molecular requirements for neutrophil influx into joints become more permissive after inflammation is initiated.

Protein tyrosine kinase p56-Lck regulates lymphocyte function-associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell.

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL-2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function-associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA-ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2-Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers.

In order to analyze whether measles virus (MV) is transported via transmigrating leukocytes across endothelial barriers or whether virus spreads via infection of endothelial cells and basolateral release, we investigated the migratory behavior of infected human primary T lymphocytes across polarized cell layers of human brain microvascular endothelial cells. We found that the capacity of lymphocytes to migrate through filter pores was only slightly affected by wild-type MV infection, whereas their capacity to migrate through endothelial barriers was drastically reduced. MV infection stimulated the expression and activation of the leukocyte integrins LFA-1 and VLA-4, mediating a strong adherence to the surface of endothelial cells. Furthermore, the formation of engulfing membrane protrusions by endothelial cells, so-called transmigratory cups, was induced, but transmigration was impaired. As a consequence of this close cell-cell contact, MV infection was transmitted from lymphocytes to the endothelium. MV envelope proteins were expressed on the apical and basolateral surfaces of infected polarized endothelial cells, and virus was released from both sides. Wild-type MV infection did not induce the formation of syncytia, suggesting virus spread from cell to cell via cell processes and contacts. Our data indicate that transendothelial migration of infected T cells is strongly inhibited, whereas virus can cross endothelial barriers by productive infection of the endothelium and subsequent bipolar virus release.

Qualitative and quantitative characteristics of rotavirus-specific CD8 T cells vary depending on the route of infection.

CD8 T-cell response provides an important defense against rotavirus, which infects a variety of systemic locations in addition to the gut. Here we investigated the distribution, phenotype, and function of rotavirus-specific CD8 T cells in multiple organs after rotavirus infection initiated via the intranasal, oral, or intramuscular route. The highest level of virus-specific CD8 T cells was observed in the Peyer's patches of orally infected mice and in the lungs of intranasally infected animals. Very low levels of virus-specific CD8 T cells were detected in peripheral blood or spleen irrespective of the route of infection. Rotavirus-specific CD8 T cells from Peyer's patches of orally infected mice expressed high levels of CCR9, while CXCR6 and LFA-1 expression was associated with virus-specific CD8 T cells in lungs of intranasally infected mice. Oral infection induced the highest proportion of gamma interferon(-) CD107a/b(+) CD8 T cells in Peyer's patches. When equal numbers of rotavirus-specific CD8 T cells were transferred into Rag-1 knockout mice chronically infected with rotavirus, the donor cells derived from Peyer's patches of orally infected mice were more efficient than those derived from lungs of intranasally infected animals in clearing intestinal infection. These results suggest that different routes of infection induce virus-specific CD8 T cells with distinct homing phenotypes and effector functions as well as variable abilities to clear infection.

Identification of a NCR+/NKG2D+/LFA-1(low)/CD94(-) immature human NK cell subset.

CD56(bright) natural killer (NK) cells, generated in vitro from CD34+ hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56(bright) cells expressed CD117, CD25, natural cytotoxicity receptors (NCRs), NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56(bright)/NCRs+/NKG2D+/LFA-1(-)/CD94(-) subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34(-)/LFA-1(low)/NKp46(dim)/NKG2D(dim)/CD94(-) subset that resembled an immature stage of NK cells present in lymph nodes. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of NK cell differentiation.

Presentation of integrins on leukocyte microvilli: a role for the extracellular domain in determining membrane localization.

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha4beta7, alphaLbeta2, and alphaMbeta2 were heterologously expressed in K562 cells. alpha4beta7 initiated adhesion under flow and localized to microvilli, whereas beta2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha4beta7 cytoplasmic and/or transmembrane domains with the homologous domains of alphaLbeta2 or alphaMbeta2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha4beta7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

Increased expression of cell adhesion molecules in inflammatory myopathies: diagnostic utility and pathogenetic insights.

CONTEXT: Idiopathic inflammatory myopathies (IMs) have been postulated to be of autoimmune origin on the basis of expression of markers like MHC-1 and other mediators involved in autoimmunity such as cell adhesion molecules. AIMS: The present study aims to analyze the expression of cell adhesion molecules ICAM-1 and VCAM-1 and their respective ligands LFA-1 and VLA-4 in IMs, and to assess whether these markers, besides MHC-class 1 antigen and membrane attack complex (MAC), could be of any help in the diagnosis of these diseases. MATERIAL AND METHODS: Retrospective analysis of 119 muscle biopsies consisting of 55 IMs (21 dermatomyositis, 31 polymyositis and 3 inclusion body myositis) and 64 controls received in our department from January 2004 to December 2005 was carried out immunohistochemically using monoclonal antibodies. STATISTICAL ANALYSIS: Chi square test and test for validity were used for analysis of differences in expression. RESULTS: Expression of ICAM and VCAM was significantly upregulated on blood vessels and muscle fibres in IMs as compared to controls, in which expression was weak or absent. LFA and VLA-4 were expressed in inflammatory cells in all inflammatory diseases in almost equal numbers. CONCLUSIONS: IMs comprise 6% of all muscle diseases and IBM is not a common IM in India as reported in the Western literature. Our findings support the hypothesis of autoimmune origin of IMs. The difference between expression of these molecules in IMs and controls also has diagnostic implications and these markers should be included along with MHC-1 antigen and membrane attack complex (MAC) in the existing diagnostic armamentarium.

Glomerular endothelium in kidneys with congenital nephrotic syndrome of the Finnish type (NPHS1).

BACKGROUND: The role of glomerular capillary endothelium in the pathophysiology of nephrotic kidney diseases is poorly known. We analysed the glomerular endothelial lesions in kidneys from patients with congenital nephrotic syndrome of the Finnish type (NPHS1). The disorder is caused by a genetic defect in a major podocyte slit diaphragm protein, nephrin. It manifests as nephrotic syndrome soon after birth and leads to glomerular sclerosis in early childhood. METHODS: The glomerular capillary and endothelial cell lesions in NPHS1 kidneys nephrectomized at infancy were studied by electron and light microscopy, immunohistochemistry and cytokine antibody array. RESULTS: Mesangial expansion and capillary obliteration were evident in practically all NPHS1 glomeruli. No thrombus formation was detected by fibrin staining. Electron microscopy revealed endothelial blebs (endotheliosis). The endothelial fenestration and the attachment of endothelial cells to the basement membrane were, however, quite normal. This fits to the abundant expression of a vascular endothelial growth factor (VEGF) and its transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), in NPHS1 glomer- uli. The proliferative activity of the intracapillary cells was modest and no apoptosis was detected. The expression of an endothelial adhesion molecule, intercellular adhesion molecule 1 (ICAM-1) and several chemokines was upregulated in NPHS1 glomeruli as compared to adult control kidneys. The recruitment of leukocytes carrying ligands for the major endothelial adhesion molecules, however, was modest in the mesangial area of NPHS1 glomeruli. CONCLUSIONS: The findings indicate that the glomerular endothelium is quite resistant to the nephrotic state in NPHS1 kidneys and underscores the importance of mesangial cells in the progression of glomerular sclerosis.

Role of SPA-1 in phenotypes of chronic myelogenous leukemia induced by BCR-ABL-expressing hematopoietic progenitors in a mouse model.

SPA-1 is a negative regulator of Rap1 signal in hematopoietic cells, and SPA-1-deficient mice develop myeloproliferative disorders (MPD) of long latency. In the present study, we showed that the MPDs in SPA-1(-/-) mice were associated with the increased hematopoietic stem cells expressing LFA-1 in bone marrow and their premature mobilization to spleen with extensive extramedullary hematopoiesis, resembling human chronic myelogenous leukemia (CML). We further showed that human BCR-ABL oncogene caused a partial down-regulation of endogenous SPA-1 gene expression in mouse hematopoietic progenitor cells (HPC) and immature hematopoietic cell lines. Although both BCR-ABL-transduced wild-type (wt) and SPA-1(-/-) HPC rapidly developed CML-like MPD when transferred to severe combined immunodeficient mice, the latter recipients showed significantly increased proportions of BCR-ABL(+) Lin(-) c-Kit(+) cells compared with the former ones. Serial transfer experiments revealed that spleen cells of secondary recipients of BCR-ABL(+) wt HPC failed to transfer MPD to tertiary recipients due to a progressive reduction of BCR-ABL(+) Lin(-) c-Kit(+) cells. In contrast, SPA-1(-/-) BCR-ABL(+) Lin(-) c-Kit(+) cells were sustained at high level in secondary recipients, and their spleen cells could transfer MPD to tertiary recipients, a part of which rapidly developed blast crisis. Present results suggest that endogenous SPA-1 plays a significant role in regulating expansion and/or survival of BCR-ABL(+) leukemic progenitors albeit partial repression by BCR-ABL and that Rap1 signal may represent a new molecular target for controlling leukemic progenitors in CML.