Biomedical subjects

L B Klickstein

Publications and source records attributed to L B Klickstein.

At least 19 recordsLinked to original sources

PubMed2005

A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1).

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.

Animals↗

PubMed2001

A dominant Jurkat T cell mutation that inhibits LFA-1-mediated cell adhesion is associated with increased cell growth.

LFA-1 exists in a low avidity state on resting leukocytes and is believed to adopt a high avidity state when the cells are exposed to a stimulus. Current evidence supports both aggregation of LFA-1 on the cell surface and conformational changes in the reversible acquisition of a high avidity state. We studied this regulation by selecting a Jurkat T cell clone, J-lo1.3, that expresses LFA-1 yet fails to bind to purified ICAM-1 despite treatment of the cells with PMA or Mn2+. Several lines of evidence demonstrated the absence of any changes within LFA-1 itself. LFA-1 protein purified from the J-lo1.3 clone and the wild-type Jurkat clone, Jn.9, were found to be functionally equivalent. The cDNA sequences encoding the LFA-1 alpha- and beta-chains from J-lo1.3 were identical with the published sequences except for nine base pairs. However, these differences were also found in a Jurkat mutant with a constitutively avid phenotype, J+hi1.19 or the wild-type Jn.9 genomic or cDNA. Fusion of J-lo1.3 with Jn.9 yielded hybrids that exhibited the J-lo1.3 adhesion phenotype, which indicated a dominant mutation in J-lo1.3. This phenotype was relatively specific for LFA-1 among all integrins expressed by Jurkat. Interestingly, the J-lo1.3 cells had a 1.2-fold faster doubling time than did the Jn.9 cells. Reversion of J-lo1.3 to the wild-type adhesion phenotype by mutagenesis and selection also decreased the growth rate. These data support a connection between cellular growth and cellular adhesion in lymphocytes.

Animals↗

PubMed2001

gp49B1-alpha(v)beta3 interaction inhibits antigen-induced mast cell activation.

We have identified the integrin alpha(v)beta3 as a ligand for mouse gp49B1, thus identifying a new class of ligand for a member of the family of inhibitory immunoreceptors that bear C2-type immunoglobulin-like domains. The specific interaction was shown by both cell-protein and cell-cell binding assays. In addition, we found that the interaction of alpha(v)beta3 with gp49B1 on bone marrow-derived mouse mast cells inhibited antigen-induced immunoglobulin E-mediated cell activation. Because neither gp49B1 nor alpha(v)beta3 exhibit substantive allelic variation, their newly appreciated interaction may reflect an innate pathway for down-regulating the activity of mast cells.

Animals↗

PubMed2001

Lack of age-associated LFA-1 up-regulation and impaired ICAM-1 binding in lymphocytes from patients with Down syndrome.

To investigate the role of LFA-1 in the immune defects in DS patients, we analysed lymphocytes from DS patients in LFA-1 expression and LFA-1 mediated cell adhesion. DS patients less than 2 years of age expressed a higher level of LFA-1 when compared with age-matched controls. The difference in LFA-1 expression was much less significant in older DS patients when compared with age-matched children. Although older children (2-15-year-old groups) without DS tend to increase their expression of lymphocyte LFA-1 when compared with younger normal children (0-2 years old), DS patients showed no age-associated increase in lymphocyte LFA-1 expression. Two-colour analysis with CD4/CD8 and LFA-1 in patients and controls showed that proportions of CD4 + lymphocytes were comparable in DS patients and controls, while the proportion of CD8 + lymphocytes was higher in older DS patients. Expression levels of LFA-1 on both CD4 + and CD8 + lymphocytes in younger DS patients were higher when compared with age-matched controls and close to the expression levels in the older DS group. Proportions of memory lymphocytes expressing the CD45RO isoform were higher in both younger and older DS patients when compared with age-matched control groups. Noticeably, the LFA-1 expression levels on CD45RO lymphocytes from younger DS patients were higher than the levels of the controls and declined in the older DS group. We tested lymphocytes (EBV transformed B cells, resting and anti-CD3 stimulated T cells) for cellular adhesion to recombinant ICAM-1 and found that lymphocytes from DS patients were less adhesive, even though their beta2 integrin expression was comparable with that of normal controls. These results suggest that more generalized pathological processes, such as early senescence of the immune system or ineffective lymphocyte activation, and subsequent integrin dysfunction may underlie the immune defects in DS patients.

Adolescent↗

PubMed2001

Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1alpha: implications for leukocyte transmigration.

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.

Cell Movement↗

PubMed2001

Dissection of the temporal artery in a patient with giant cell arteritis.

A 74-year-old woman presented to her rheumatologist with classic symptoms of giant cell arteritis. The temporal arteries were strikingly swollen, warm, and erythematous. On biopsy of the right temporal artery, a focal dissection was found associated with a pan-arteritis and giant cells. Isolated temporal artery dissection in giant cell arteritis has not been reported previously. We propose that the unusually intense vascular inflammation may have weakened the vessel wall, so that the dissection occurred during the routine physical exam or biopsy. We believe this case illustrates that physicians should take special care in the examination of floridly inflamed vessels, because vigorous palpation might lead to dissection. In the case of patients with giant cell arteritis, dissection may result in an increased risk of ischemic complications, such as scalp necrosis.

Journal Article↗

PubMed2000

Complement receptor 1/CD35 is a receptor for mannan-binding lectin.

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.

Binding Sites↗

PubMed2000

Intercellular adhesion molecule 1 and beta2 integrins in C1q-stimulated superoxide production by human neutrophils: an example of a general regulatory mechanism governing acute inflammation.

OBJECTIVE: To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins in the production of superoxide (O2-) by C1q-stimulated human polymorphonuclear leukocytes (PMN). METHODS: PMN were pretreated with F(ab')2 fragments of monoclonal antibodies (mAb) that blocked or did not block beta2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O2- was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1. RESULTS: Blocking mAb to the shared beta2 integrin subunit, CD18, completely inhibited the O2- response triggered by immobilized C1q, while blocking mAb to the alpha subunits of the beta2 integrins each partially blocked the O2- response. PMN treated with C1q were found to activate the beta2 integrins lymphocyte function-associated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O2- production by PMN. CONCLUSION: beta2 integrin binding to an ICAM provided an essential costimulatory signal for O2-production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O2- production: a first signal that also activates PMN beta2 integrins, followed by a second, beta2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O2- would serve as a regulatory mechanism to limit the production of O2- to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing up-regulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor alpha, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.

Antibodies, Blocking↗

PubMed2000

C5a-stimulated human neutrophils use a subset of beta2 integrins to support the adhesion-dependent phase of superoxide production.

Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual beta2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other beta2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for alphad/beta2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase of-superoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and alphad/beta2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.

Antibodies, Monoclonal↗

PubMed1999

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.

Antigen-Antibody Complex↗

PubMed1999

C1q-binding proteins and C1q receptors.

Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.

Carrier Proteins↗

PubMed1999

Specific activation of leukocyte beta2 integrins lymphocyte function-associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains.

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains.

CD18 Antigens↗

PubMed1998

Chemokine-induced monocyte transmigration requires cdc42-mediated cytoskeletal changes.

The ras-related small GTPases of the rho family coordinate the assembly of complex cytoskeletal structures crucial for cell motility and polarization. Cdc42 controls filopodia formation in some cell types; however, other physiological functions, agonists and subsequent signaling cascades remain to be elucidated. Expression of cdc42 mutants in monocytic cells showed that CC chemokines regulate the rearrangement of the actin cytoskeleton via cdc42, i.e. formation of filopodia-like projections was induced by CC chemokines or dominant active cdc42, while dominant inactive cdc42 prevented its stimulation with CC chemokines. Both cdc42 mutants inhibited CC chemokine-induced monocyte migration across bare filters or across filters coated with endothelium, implicating cdc42 activity and its effector functions in chemotaxis. In contrast, cdc42 mutants did not affect the rho-dependent activation integrin avidity by CC chemokines. The chemokine-induced signaling pathways involved phosphoinositide 3-kinase upstream of cdc42, as shown by inhibition of cytoskeletal reorganization with wortmannin. These data identify CC chemokines as physiological agonists of cdc42 and reveal its functional importance in chemotaxis and extravasation of monocytes.

Actins↗

PubMed1997

Characterization of lymphocyte function-associated antigen 1 (LFA-1)-deficient T cell lines: the alphaL and beta2 subunits are interdependent for cell surface expression.

The leukocyte, or beta2, integrins are heterodimeric cell surface molecules that share a common beta subunit, and have unique alpha subunits. LFA-1 is the predominant beta2 integrin on lymphocytes, and plays an important role in many lymphocyte functions; however, most studies of the cytoplasmic regions of LFA-1 have been performed in transfected epithelial cells, such as COS, in part because no lymphoid cell lines deficient in the LFA-1 alpha subunit have been described. To address structure-function studies of beta2 integrins in relevant cell types, two T lymphoblastoid cell clones that completely lack cell surface LFA-1, J-(beta2).7 and SK-(beta2).7, derived from Jurkat and SKW3, respectively, were prepared by chemical mutagenesis and selection. Biosynthetic labeling and immunoprecipitation showed that the J-(beta2).7 clone did not translate any LFA-1 alpha subunit protein, while the SK-(beta2).7 cells did not synthesize any beta2 subunit protein. Northern blot analysis of poly(A+) RNA from these cells revealed an absence of the corresponding mRNA in each case. By transfection analysis, only the alpha subunit reconstituted LFA-1 expression in the J-(beta2).7 cells, while only the beta subunit restored cell surface LFA-1 expression in the SK-(beta2).7 cells. Functional studies with the parental cell lines, the J-(beta2).7 and SK-(beta2).7 cells, and the transfectants showed that all binding of Jurkat and SKW3 cells to purified ICAM-1 is mediated by LFA-1, and the reconstituted LFA-1 expressed by the J-(beta2).7 and SK-(beta2).7 transfected cells is regulated normally.

CD18 Antigens↗

PubMed1997

Complement receptor type 1 (CR1, CD35) is a receptor for C1q.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Binding Sites↗

PubMed1996

Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen 1 (LFA-1).

Intercellular adhesion molecule 3 (ICAM-3; CD50) is the predominant counter-receptor on resting T cells and monocytes for the leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), and may play an important role in the initial stages of the T cell-dependent immune response. Deletion of individual immunoglobulin superfamily (IgSF) domains of ICAM-3 and ICAM-3 IgSF domain chimeras with CD21 showed there is a single LFA-1 binding site in ICAM-3 and that IgSF domain 1 is necessary and sufficient for LFA-1 binding. Epitope mapping and functional studies performed with 17 anti-ICAM-3 monoclonal antibodies demonstrated that only some monoclonal antibodies, with epitopes wholly within domain 1 of ICAM-3, were able to block binding of ICAM-3 bearing cells to purified LFA-1, in agreement with the data obtained from the domain deletion mutants and CD21 chimeras. Analysis of a panel of 45 point mutants of domain 1 of ICAM-3 identified five residues that may contact LFA-1 as part of the binding site, Asn23, Ser25, Glu37, Phe54, and Gln75. These five residues are predicted by molecular modeling, based on the structure of vascular cell adhesion molecule 1 (VCAM-1), to cluster in two distinct locations on domain 1 of ICAM-3 on the BED face (Asn23 and Ser25) and on the C strand or CD loop (E37), the E strand (F54), and the FG loop (Q75). The residues, Asn23 and Ser25, comprise a consensus N-linked glycosylation site.

Amino Acid Sequence↗

PubMed1993

Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes.

The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.

Antigens, CD↗

PubMed1993

Cloning and expression of intercellular adhesion molecule 3 reveals strong homology to other immunoglobulin family counter-receptors for lymphocyte function-associated antigen 1.

Based on protein sequence, we have isolated a cDNA for intercellular adhesion molecule 3 (ICAM-3), the most recently defined counter-receptor for lymphocyte function-associated antigen 1 (LFA-1). Expression of the cDNA yields a product that reacts with monoclonal antibody to ICAM-3 and functions as a ligand for LFA-1. The deduced 518-amino acid sequence of the predicted mature protein defines a highly glycosylated type I integral membrane protein with five immunoglobulin (Ig)-like domains. The five Ig-like domains of ICAM-3 are highly homologous with those of human ICAM-1 (52% identity) and human ICAM-2 (37% identity).

Amino Acid Sequence↗