Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.     

Patching and capping of LFA-1 molecules on human lymphocytes

Summary The distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37°C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompained by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.     

Patching and capping of LFA-1 molecules on human lymphocytes.

The distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37 degrees C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompanied by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.     

Deficiency of two human leukocyte surface membrane glycoproteins (Mo1 and LFA-1)

Structural and functional features of a novel disorder characterized by recurrent bacterial infections are reviewed. This disease is associated with a number of phagocyte adhesion defects. In 10 patients, phenotypic analysis with monoclonal antibodies (MAb) revealed the same basic defect in all patients: deficiency of at least two leukocyte surface glycoproteins, Mo1 and LFA-1. These two antigens have distinct alpha subunits (Mo1 alpha = 155 kilodaltons, LFA-1 alpha = 177 kilodaltons) noncovalently linked to a common beta subunit (94 kilodaltons). Mo1 is closely associated with or identical to a receptor for the iC3b fragment of the third component of complement. LFA-1 is involved in lymphocyte proliferation, cytotoxicity, and natural killing. MAb directed to this family of glycoproteins induce functional defects in normal cells similar to those observed in deficient cells. In normal cells, the surface expression of these glycoproteins is regulated by the state of cell activation. Mitogens and alloantigens significantly increase the surface expression of LFA-1 on T lymphocytes. Stimuli that induce degranulation in neutrophils increase the surface expression of Mo1. In all patients with combined Mo1, LFA-1 deficiency, the predominant clinical manifestations were more characteristic of a phagocyte than a lymphocyte disorder. In vitro studies, however, reveal significant defects in phytohemagglutinin-induced proliferation that are more apparent at lower concentrations of the lectin. In some families, more than one sibling is affected. Intermediate levels of Mo1 were observed on granulocytes from both parents of one child. In one family, however, only the mother had significantly reduced levels of Mo1, which indicates heterogeneity in the inheritance of this disorder.     

Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1, and two isoforms of LFA-3

Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.     

Immunologic abnormalities in a patient with multiple neoplasms.

Pernicious anemia, antithyroid antibodies, IgG(K) M component, depressed production of other immunoglobulins and impaired function of peripheral blood lymphocytes developed in a patient with adenocarcinoma of the colon, transitional-cell carcinoma of the tongue and clear-cell parathyroid adenoma. This case demonstrates that immunologic abnormalities and multiple malignant neoplasms may coexist in one patient. It lends support to the hypothesis that neoplastic processes and autoimmunity may both be related to impaired immune surveillance.     

Electrophoretic study of lymphocytes from a patient with hairy cell leukemia

The electrophoretic mobility of lymphocytes is identified in the peripheral blood and from the spleen taken from a patient with hairy cell leukemia. Lymphocytes reveal an unimodal distribution with a low mobility in the histogram. There are no differences between the results obtained by investigating the peripheral blood and those of the spleen. Hairy cells could be delimited from lymphocytes of chronic lymphatic leukemia and immunocytoma by determining EPM.     

Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion

The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

Cutting edge: expression of IL-1 receptor-associated kinase-4 (IRAK-4) proteins with mutations identified in a patient with recurrent bacterial infections alters normal IRAK-4 interaction with components of the IL-1 receptor complex

In a patient with recurrent bacterial infections and profound hyporesponsiveness to LPS and IL-1, we previously identified two mutations in IL-1R-associated kinase-4 (IRAK-4) that encoded proteins with truncated kinase domains. Overexpression of either of these mutant IRAK-4 variants in HEK293 cells failed to activate endogenous IRAK-1 and suppressed IL-1-induced IRAK-1 kinase activity, in contrast to wild-type (WT) IRAK-4. In this study, interactions of WT and mutant IRAK-4 species with IL-1R, IRAK-1, and MyD88 in HEK293 transfectants were compared. IL-1 induced a strong interaction among the IL-1R, activated IRAK-1, MyD88, and WT, but not mutant, IRAK-4. Truncated IRAK-4 proteins constitutively interacted more strongly with MyD88 and blunted IL-1-induced recruitment of IRAK-1 and MyD88 to the IL-1R. Thus, decreased IL-1-induced association of IRAK-1 and MyD88 with the IL-1RI may result from sequestration of cytoplasmic MyD88 by IRAK-4 mutant proteins. Therefore, mimetics of these truncated IRAK-4 proteins may represent a novel approach to mitigating hyperinflammatory states.     

Lymphocyte function-associated antigens one (LFA-1) on B and on T lymphocytes bind a monoclonal antibody with different affinities

The kinetics of the binding of an anti-LFA-1 monoclonal antibody to lymphocytes of the B and T lineages was studied. A Kd approximately ten times lower was observed when binding the antibody to B splenocytes compared to T splenocytes. Using Fab fragments and measuring the dissociation rate constants gave a similar difference in binding kinetics of anti-LFA-1 between the different splenocytes. This difference of cell-binding affinity was independent of the state of stimulation of the cells by lipopolysaccharide or concanavalin A. Thymocytes reacted with the antibody with the same Kd as T splenocytes. The combined results indicate that LFA-1 from B and from T lymphocytes have a different conformation. Binding with a higher affinity to B and a lower affinity to T lymphocytes could also be demonstrated using liposomes coated with anti-LFA-1 antibody.     

Immunologic and structural studies of lipopolysaccharides from Pseudomonas cepacia

O-serotyping of 30 Pseudomonas cepacia strains isolated from the soil and rhizosphere of different plant species in the territory of the USSR has been performed using 15 O-typing antisera according to the Heidt and Nakamura schemes. It is suggested to introduce two new O-serogroups (serogroups K and L) into the available P. cepacia classification scheme. They are most often met among the P. cepacia strains in different geographical areas of the USSR simultaneously with serogroups 2 (G) and 1 (D). To elucidate the molecular principles of serological inhomogeneity of the species the immunochemical studies of lipopolysaccharides of a number of P. cepacia strains have been conducted and the structure has been determined for repeating links of O-specific polysaccharides of P. cepacia strains attributed to 4 Nakamura serogroups, 3 Heidt serogroups, to serogroups K and L, as well as for certain strains from the collection of the Institute of Microbiology and Virology of the Ukr. SSR Academy of Sciences.     

Hypoxia and HIF-1 activation in bacterial infections

For most of the living beings, oxygen is one of the essential elements required to sustain life. Deprivation of oxygen causes tissue hypoxia and this severely affects host cell and organ functions. Tissue hypoxia is a prominent microenvironmental condition occurring in infections and there is a body of evidence that hypoxia and inflammation are interconnected with each other. The primary key factor mediating the mammalian hypoxic response is hypoxia inducible factor (HIF)-1, which regulates oxygen homeostasis on cellular, tissue and organism level. Recent studies show that HIF-1 plays a central role in angiogenesis, cancer and cardiovascular disease but also in bacterial infections. Activation of HIF-1 depends on the nature of the pathogen and the characteristics of infections in certain hosts. Up to date, it is not completely clear whether the phenomenon of HIF-1 activation in infections has a protective or detrimental effect on the host. In this review, we give an overview of whether and how hypoxia and HIF-1 affect the course of infections.     

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion.

Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.     

The relationship between mucosal immunoresponse and clinical outcome in patients with recurrent upper respiratory tract infections treated with a mechanical bacterial lysate

This open prospective study aims to evaluate whether a therapy with a polyvalent mechanical bacterial lysate (PMBL) could be associated to the enhancement of the locoregional immunoresponse in patients with recurrent upper respiratory tract infections. Forty patients (23 females and 17 males) were enrolled, 33 of whom concluded the study. The duration of the study was six months and each patient was visited five times. Twenty-six patients had an objective improvement in clinical and medical locoregional conditions, while in seven patients the treatment did not result in an objective amelioration. Twenty-five out of 27 patients with clinical response were characterized by an increase of specific antibodies against PMBL antigens in salivary fluids. Only two patients, with a non-significant clinical result, had a slight increase in the concentration of salivary specific IgA. The association between PMBLspecific immunoglobulin titers and clinical results was significant for IgG and IgA, but not significant for IgM. Th1 switch was detected only in patients with clinical amelioration, while the Th0 phenotype was observed in three responder and four non-responder patients. Weak Th2 polarization was also observed in one clinical responsive patient. The capacity of effectively opsonizing living bacteria was detected in samples derived from responder patients. These results suggest that PMBL treatment was able to trigger an efficient and well-targeted immune-response resulting in positive clinical outcome of the patients treated.     

Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.