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Biomedical subjects

B W McIntyre

Publications and source records attributed to B W McIntyre.

At least 37 records · Page 2Linked to original sources
PubMed1997

Changes in gravity inhibit lymphocyte locomotion through type I collagen.

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Animals↗
PubMed1996

Alpha 4 beta 1 (CD49d/CD29) integrin costimulation of human T cells enhances transcription factor and cytokine induction in the absence of altered sensitivity to anti-CD3 stimulation.

The integrin alpha 4 beta 1 can provide a costimulus to induce IL-2 secretion and IL-2R expression leading to enhanced proliferation of purified, peripheral blood T cells. Similar to expression of IL-2, we demonstrated that recombinant vascular-cell adhesion molecule-1, when co-immobilized with anti-CD3 mAb, significantly enhanced the induction of transcription factors NF-AT, AP-1, and NF-kappa B as determined by electromobility shift assays. alpha 4 beta 1 ligation alone had no effect on transcription factor binding. The requirements for induction of transcription factors reflected the requirements for the secretion of multiple cytokines, including IL-2, TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF. In contrast to freshly isolated T cells, in vitro-cultured T cells did not require costimulation for cytokine secretion in response to anti-CD3 alone. Comparison of the dose response to anti-CD3 stimulation demonstrated that half-maximal induction of IL-2 was achieved using the same dose of anti-CD3 for both freshly isolated and cultured T cells. Furthermore, the dose of OKT3 required to achieve half-maximal activation was the same using PMA or different concentrations of alpha 4 beta 1 ligands. Therefore, costimulation by alpha 4 beta 1 ligands was not due to stabilization of the interaction of the cells with its substrate. We conclude, rather, that alpha 4 beta 1 in freshly isolated T cells delivers a distinct signal that synergizes early with signals initiated by TCR/CD3 ligation to induce DNA binding of multiple transcription factors required for cytokine gene induction.

CD3 Complex↗
PubMed1996

Specific inhibition of T lymphocyte coactivation by triggering integrin beta 1 reveals convergence of beta 1, beta 2, and beta 7 signaling pathways.

T cell coactivation is a dynamic process subject to integrin-dependent positive and negative regulation. Costimulation of human peripheral blood T cells by CD3 mAb OKT3 in conjunction with anti-alpha 4 has been shown to be down-regulated by the anti-beta 1.1 epitope-specific mAb 18D3. As expected, maximal costimulation induced by alpha 4-specific mAb L25 was inhibited (70%) by the addition of soluble mAb 18D3. Surprisingly, soluble mAb 18D3 inhibited maximal proliferation induced by the costimulatory alpha 4 beta 7-specific mAb ACT-1 by 40%, thus demonstrating that one integrin subfamily can regulate the activity of another. To determine whether mAb 18D3 could regulate more than alpha 4-associated integrin-mediated costimulation, non-alpha 4 integrins were tested. mAb 18D3 inhibited maximal proliferation induced by alpha 4-specific mAb 3D6, and an alpha 4-specific mAb 16. This clearly demonstrates that a variety of integrin costimulatory molecules (of the beta 1, beta 2, and beta 7 subfamilies) can be regulated negatively by mAb 18D3. To analyze the specificity of this negative regulation, other cell surface costimulatory molecules were tested for susceptibility to mAb 18D3. Although Abs specific for CD4, CD26, CD28, CD44, CD45RA, or CD45RO were sufficient to activate T cells when co-immobilized with anti-CD3 mAb, all were refractory to the inhibitory effects of mAb 18D3. Inhibition of T cell activation directly correlated with diminished IL-2 production. This suggests that mAb 18D3 selectively regulates integrin-dependent T cell activation by delivering a negative effect at some common point utilized by various integrin subfamilies.

Antibodies, Monoclonal↗
PubMed1996

ADP-ribosylation of the G protein Rho inhibits integrin regulation of tumor cell growth.

Using a gastric derived tumor line, we investigated the involvement of beta 1 integrin and Rho in cell growth regulation in response to collagen. The addition of C3 exoenzyme from clostridium botulinum to specifically ribosylate and inhibit the function of the rho gene products inhibited cellular proliferation in a dose-dependent fashion. C3 exoenzyme exhibited broad cytostatic activity toward a number of tumor lines and induced G0/G1 accumulation, cyclin A inhibition, and pronounced alterations in cell morphology. Integrin-mediated adhesion to collagen led to the expression of the cyclin A gene whose expression could be blocked using anti-beta 1 integrin monoclonal antibodies. Phospholipid levels were induced upon beta 1 integrin-mediated adhesion to collagen, and the phospholipid induction was inhibited by either antibodies to beta 1 integrin or pretreatment of cells with C3 exoenzyme. Significant reduction in phospholipid levels correlated with proliferation for a panel of tumor lines deprived of adhesion to substrate. These results implicate a novel role for integrins and Rho in the regulation of tumour growth in response to matrix.

ADP Ribose Transferases↗
PubMed1996

Induction of homotypic lymphocyte aggregation: evidence for a novel activation state of the beta1 integrin.

Intercellular adhesion of Jurkat lymphocytic cells was investigated by use of monoclonal antibodies 33B6 and 18D3, which bind to the beta1 integrin receptor. 33B6 induced homotypic aggregation of Jurkat cells, whereas 18D3 inhibited this aggregation. Jurkat cells could he induced to aggregate at low 33B6 concentrations corresponding to 5% beta1 integrin site occupancy, and the rate of aggregation was maximum at 30% occupancy. Simultaneous addition of mAb 18D3 and 33B6 demonstrated that the two antibodies mediate changes in the beta1 integrin activation state that are competitive in nature. Aggregation through beta1 integrin induced by 33B6 was reversed by subsequent addition of 18D3. To further examine the mechanism by which 33B6 and 18D3 affect cell adhesion function, we explored the binding of monoclonal antibody (mAb) 15/7. This mAb recognizes an activation epitope of the beta1 integrin and has been shown to sustain cell adhesion to vascular cell adhesion molecule 1 (VCAM-1) and fibronectin. Activation of Jurkat cells with Mn2+ caused a 2.5-fold increase in 15/7 binding but did not increase binding of 33B6. 33B6 partially blocked 15/7 binding to beta1 integrin on unstimulated and Mn2+-activated Jurkat cells. 18D3 did not affect mAb 15/7 binding. These results indicate that 33B6 and 18D3 modulated homotypic aggregation by inducing a novel activation state of the very late activation integrin distinct from the state recognized by 15/7, which supports cell binding to VCAM-1 and fibronectin.

Antibodies, Monoclonal↗
PubMed1995

Regulation of lymphocyte pseudopodia formation by triggering the integrin alpha 4/beta 1.

Video microscopy and digital imaging were used to quantitatively analyze lymphocyte adhesion and formation of pseudopodia on the extracellular matrix protein fibronectin (FN). A morphology kinetics assay comparing pseudopodial extension values over a 24-h period showed that HPB-ALL T leukemic cells undergo a wave of morphologic change, returning to a round shape after 8 h. Using anti-alpha 4 and anti-alpha 5 mAbs and a panel of cell types that are single or double positive for expression of the alpha 4/beta 1 and alpha 5/beta 1 FN binding integrins, it was determined that cell adhesion to FN was influenced by both beta 1-integrins, whereas alpha 4/beta 1 was found to be the major FN receptor mediating pseudopodia extension. The protein kinase inhibitor staurosporine, the protein kinase C inhibitors calphostin C and chelerythrine, and the protein tyrosine kinase inhibitor herbimycin A blocked pseudopodial extension in HPB-ALL cells. In contrast, two cAMP-dependent protein kinase inhibitors H8 and H89 did not inhibit. Inhibitors of phospholipase A2, lipoxygenases, and cyclooxygenases could block formation of pseudopodia, yet had little or no effect on cell adhesion to FN. The preincubation of cells with arachidonic acid could prevent the inhibition mediated by the reversible phospholipase A2 inhibitor cibacron blue. We conclude that the formation of lymphocyte pseudopodia in response to FN can utilize the adhesive and signaling activities of the alpha 4/beta 1-integrin and the enzymatic activities of protein kinases and phospholipases.

Antibodies, Monoclonal↗
PubMed1995

Novel tumor-associated accessory molecules involved in the gamma/delta cytotoxic T-lymphocyte-Burkitt's lymphoma interaction.

BACKGROUND: Tumor specific cytotoxic T lymphocytes (CTL) recognize antigen via the T-cell receptor (TCR). In addition, recognition requires accessory molecules involved in adhesion and signal transduction. The authors previously have characterized an autologous, Burkitt's lymphoma specific CTL line that uses the gamma-delta TCR to recognize antigen in a nonclassical context. The current study was undertaken to identify novel accessory molecules involved in this unusual TCR-tumor cell interaction. METHODS: A panel of monoclonal antibodies was generated against a Burkitt's lymphoma cell line and was screened for inhibition of autologous, tumor specific, cytolysis by a gamma-delta CTL line. Proteins identified by these monoclonal antibodies were further characterized by fluorescent-activated cell sorter analysis, Western blot and immunoprecipitation. RESULTS: Three known (CD5, CD43, and CD11a/CD18) and three novel (BAM-1, BAM-2, and BAM-3) cell surface molecules involved in the gamma-delta CTL-Burkitt's lymphoma interaction were identified and characterized. CONCLUSIONS: This study identifies and provides a preliminary characterization of three novel Burkitt's lymphoma-associated molecules involved in the gamma-delta CTL-tumor cell interaction and demonstrates that CD5, CD43, and CD11a/CD18 are involved in this interaction. It is likely that other unidentified accessory molecules are also involved in this and other effector cell-tumor interactions. Identification of such molecules may be useful in the design of new immunotherapeutic approaches.

Animals↗
PubMed1995

Monoclonal antibodies that block the activity of leukocyte function-associated antigen 1 recognize three discrete epitopes in the inserted domain of CD11a.

The epitopes recognized by eight independently isolated monoclonal antibodies to the alpha chain of human and murine leukocyte function-associated antigen 1 (LFA-1), all able to inhibit receptor function, were identified. Initial localization of epitopes was accomplished using chimeric proteins constructed by splicing fragments of cDNAs encoding the alpha subunit of LFA-1 (CD11a) and the alpha subunit of the closely related leukocyte integrin, Mac-1 (CD11b). Antibody binding to CD11a/CD11b chimeras, expressed in the 293 human kidney cell line, demonstrated that the epitopes recognized by six monoclonal antibodies to human CD11a were located in a approximately 200-amino acid sequence found in all beta 2-integrin alpha subunits, termed the inserted (I) domain. Three distinct epitopes within the I domain (IdeA, IdeB, and IdeC) were identified using a series of mutants in which sequences from murine CD11a were substituted into human CD11a. A series of mutants incorporating single amino acid substitutions was used to identify individual amino acids essential for antibody binding. The location of these residues accounts for the binding specificity of LFA-1-blocking antibodies and identifies particular conserved sequences (residues 126-150) in the I domain of CD11a and homologous sequences in other beta 2-integrin alpha subunits that may be important for ligand binding.

Amino Acid Sequence↗
PubMed1995

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells.

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting beta 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to alpha 2 or beta 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.

Adenocarcinoma↗
PubMed1995

Liposomal muramyl tripeptide upregulates adhesion molecules on the surface of human monocytes.

We previously demonstrated that liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE), a biologic response modifier now undergoing phase III clinical trial in osteosarcoma, upregulated monocyte expression of several cytokines' mRNA and the subsequent production of these proteins. In the present work, we investigated whether L-MTP-PE upregulated adhesion molecules on the surface of normal human monocytes. Flow-cytometric analysis showed that several subunits of the integrins, including alpha L, alpha 5, and beta 1 subunits, and intercellular adhesion molecule-1 on the monocytes were upregulated following their stimulation with 2 micrograms/ml L-MTP-PE for 24 h. Anti-alpha L antibodies blocked monocyte-mediated tumor cell killing stimulated by L-MTP-PE. We conclude that L-MTP-PE also stimulates the increase of several molecules on the monocyte cell surface. These adhesion molecules may contribute to the increased activation of monocyte-mediated tumor cell killing seen following L-MTP-PE exposure.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1994

Identification of a combinatorial epitope expressed by the integrin alpha 4 beta 1 heterodimer involved in the regulation of cell adhesion.

The alpha 4 integrin subunit can associate with either the beta 1- or beta 7-integrin subunit to form two unique adhesion receptors alpha 4 beta 1 and alpha 4 beta 7. We developed a monoclonal antibody (mAb 19H8) that immunoprecipitated alpha 4 beta 1, induced homotypic leukocyte aggregation, and blocked the binding of cells to a synthetic peptide corresponding to the CS-1 peptide region of fibronectin. Aggregation cross-blocking analysis suggested that mAb 19H8 belonged to the group of mAbs that react with the B2 epitope of the alpha 4 subunit (alpha 4.B2 epitope); however, unlike the alpha 4.B2-specific mAb L25, mAb 19H8 did not immunoprecipitate alpha 4 beta 7. In addition, mAb 19H8 did not bind to beta 1-positive cells unless transfected with alpha 4 cDNA. These results indicated that mAb 19H8 was not specific for an individual alpha 4, beta 1, or beta 7 subunit but reacted with an epitope formed from the association of alpha 4 with beta 1. Separating the alpha 4 from the beta 1 subunit, by removing divalent cations or by treatment with high pH, disrupted mAb 19H8 binding. In contrast, the alpha 4-specific mAb L25 and the beta 1-specific mAb 18D3 could react with their respective subunits without subunit association. Therefore, mAb 19H8 defined a novel regulatory epitope expressed by the integrin alpha 4 beta 1.

Antibodies, Monoclonal↗
PubMed1994

MAb 18D3 triggering of integrin beta 1 will prevent but not terminate proliferation of human T cells.

Triggering of integrins can deliver signals that will regulate T cell activation and proliferation when coupled with TCR/CD3 signaling. While co-activation stimuli can be achieved either with immobilized natural ligands or immobilized monoclonal antibodies specific for various integrin subunits, counterposing effects can be delivered by ligation of the integrin beta 1 chain (CD29) resulting in the downregulation of T cell proliferation. Thus, integrins may play a pivotal role in cell activation and are involved in both positive and negative regulatory pathways. In this report, anti-beta 1 mAb 18D3 was used to investigate the role of beta 1 in the negative regulation of T cell proliferation. T lymphocytes were stimulated to proliferate when activated with immobilized mAb to CD3 in conjunction with all of a panel of immobilized mAb to different alpha 4 (CD49d) and beta 1 epitopes, except the anti-beta 1.1 mAb 18D3. In soluble form, mAb 18D3 inhibited the induction of DNA synthesis dependent on costimulation of CD3 and the integrin alpha 4 subunit by a mechanism independent of anti-adhesive properties. In kinetic experiments, the addition of mAb 18D3 effectively inhibited the ultimate induction of DNA synthesis at all time points until the time coinciding with the onset of T cell proliferation, indicating that triggering the beta 1.1 epitope may only act to quench activation events prior to cellular commitment to synthesize DNA. MAb 18D3 did not induce cell death nor render cells incompetent for restimulation, but appeared to selectively inhibit IL-2 synthesis with little effect on the induction of IL-2 receptor expression.

Antibodies, Monoclonal↗
PubMed1994

Integrin alpha 4 beta 7 co-stimulation of human peripheral blood T cell proliferation.

The integrin alpha 4 beta 7 mediates lymphocytes adhesion to VCAM-1 on activated endothelium, fibronectin in the extracellular matrix, and the mucosal vascular addressin MAdCAM-1. It is unclear whether alpha 4 beta 7 performs any function beyond directing specific adhesion reactions. We addressed the possibility that triggering of alpha 4 beta 7 with a specific monoclonal antibody was capable of delivering an accessory stimulus that would coactivate T cells and lead to proliferation. At submitogenic levels of anti-CD3 stimulation, triggering of alpha 4 beta 7 by immobilized mAb ACT-1 resulted in T cell blastogenesis, IL-2 production, expression of the IL-2 receptor alpha chain CD25, and ultimately DNA synthesis. These results indicate that the integrin alpha 4 beta 7 is involved in more than lymphocyte adhesion and homing but also plays a role in cell signaling.

Antibodies, Monoclonal↗
PubMed1993

Analysis of lymphocyte aggregation using digital image analysis.

We present the development and testing of a novel assay of lymphocyte adhesion based on time-resolved morphological measurements of intercellular aggregation. Homotypic lymphocyte aggregation is induced according to various protocols and monitored for several hours using video microscopy and time-lapse recording. Digital images of the aggregating cell population are acquired and analyzed to obtain the size distribution and the shape of cell aggregates. By following the temporal evolution of the size distribution of aggregates, the rates of aggregation events can be accurately quantified and compared. In addition, an analysis of the two- and three-dimensional structures of the aggregates using appropriately defined shape factors allows comparisons of mechanical binding strengths and cytoskeletal activity. To demonstrate the capabilities of the assay, we present results from a series of aggregation experiments with Jurkat cells treated with 33B6, 19H8, IC9, and 20E4 monoclonal antibodies. These monoclonal antibodies bind to various epitopes of known adhesion molecules and induce aggregation phenomena that proceed at different rates. Our results show that the assay has small repeatability error and is sensitive enough to compare aggregation events induced through distinct molecular epitopes. Used in conjunction with current biochemical detection assays and adhesion pathway modulation experiments, the developed assay will facilitate the study of cellular adhesion and aggregation mechanisms.

Antibodies, Monoclonal↗