Long-term variability of trace gases over the Indian Western Himalayan Region.

The four-year continuous measurements of CO, NOx, NH3, SO2, and O3 were carried at a high altitude site (32.12°N, 76.56°E at 1347 m AMSL) of the Indian Western Himalayan area to study the mixing ratios of these gases for understanding the changing trends of these trace gases over the region. Each of these trace gases showed significant daily and monthly variabilities. The highest variability was recorded in the monthly mean values of O3 as it varied from 10 to 63 ppb during the study period. All the trace gases except CO showed maximum variability in the pre-monsoon seasons due to the strong advection and vertical circulation of air masses at the site. The seasonal mean maxima of CO were recorded during the monsoon season, while the mean maxima of NH3 were recorded during the post-monsoon seasons. The meteorological parameters have been found to influence the mixing ratios of trace gases. The least variability in the mean seasonal mixing ratios of SO2 during the study period indicated the constant point source of SO2 near the site. The trajectories analysis revealed that the area receives maximum air masses from the southeast to the west directions where a number of the coal-based thermal power plants, industries, cement plants, and agricultural fields are also located. The influence of valley-to-mountain circulations was also observed at the site, resulting in the transport of pollutant-rich air masses from local and distant sources to the site. A comparison of the mixing ratios of different trace gases obtained in the present study is also made with the values reported for other high altitude stations in the world.

Expression and localization of insulin-like growth factor system in corpus luteum during different stages of estrous cycle in water buffaloes (Bubalus bubalis) and the effect of insulin-like growth factor I on production of vascular endothelial growth factor and progesterone in luteal cells cultured in vitro.

This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were significantly higher (P < 0.05) when the culture medium was supplemented with 100 ng/mL of IGF-I for 72 hours. Moreover, IGF-I at a dose of 100 ng/mL increased P4 and VEGF production (P < 0.05). It can be concluded that IGF family members via their autocrine and paracrine effect play significant roles in promoting angiogenesis through the production of VEGF in luteal cells and steroid synthesis through the production of key steroidogenic factors.

Dosimetric verification of a high dose rate brachytherapy treatment planning system in homogeneous and heterogeneous media.

OBJECTIVES: To verify the dosimetric accuracy of treatment plans in high dose rate (HDR) brachytherapy by using Gafchromic EBT2 film and to demonstrate the adequacy of dose calculations of a commercial treatment planning system (TPS) in a heterogeneous medium. METHODS: Absorbed doses at chosen points in anatomically different tissue equivalent phantoms were measured using Gafchromic EBT2 film. In one case, tandem ovoid brachytherapy was performed in a homogeneous cervix phantom, whereas in the other, organ heterogeneities were introduced in a phantom to replicate the upper thorax for esophageal brachytherapy treatment. A commercially available TPS was used to perform treatment planning in each case and the EBT2 films were irradiated with the HDR Ir-192 brachytherapy source. RESULTS: Film measurements in the cervix phantom were found to agree with the TPS calculated values within 3% in the clinically relevant volume. In the thorax phantom, the presence of surrounding heterogeneities was not seen to affect the dose distribution in the volume being treated, whereas, a little dose perturbation was observed at the lung surface. Doses to the spinal cord and to the sternum bone were overestimated and underestimated by 14.6% and 16.5% respectively by the TPS relative to the film measurements. At the trachea wall facing the esophagus, a dose reduction of 10% was noticed in the measurements. CONCLUSIONS: The dose calculation accuracy of the TPS was confirmed in homogeneous medium, whereas, it was proved inadequate to produce correct dosimetric results in conditions of tissue heterogeneity.

A dosimetry method in the transverse plane of HDR Ir-192 brachytherapy source using gafchromic EBT2 film.

Radiochromic film dosimetry is increasingly used in brachytherapy applications for its higher resolution ability as compared to other experimental methods. The present study was aimed to assess the accuracy and suitability of use of the improved radiochromic film model, Gafchromic EBT2, to evaluate the dose distribution in the transverse plane of microselectron HDR (192)Ir source. A specially designed and locally fabricated Polymethyl methacrylate (PMMA) phantom was used in this work for the experimental measurement of dose distribution around the source in its transverse plane. The AAPM TG-43U1 recommended radial dose function, g (r), and dose rate constant, Λ, for the source were measured using Gafchromic EBT2 film and thermoluminescent dosimeters (TLD). The EBT2 film measured dosimetric quantities were validated against their values obtained from the TLD measurements and previously published values for the same source available in literature. The dose rate constant and radial dose function for microselectron HDR (192)Ir source obtained from Gafchromic EBT2 film measurements are in agreement with their TLD measured results within 3.9% and 2.8% respectively. They also agree within the accepted range of uncertainty with their experimental and Monte Carlo calculated results reported in literature. This work demonstrates the suitability of using Gafchromic EBT2 film dosimetry in characterization of dose distribution in the transverse plane of HDR Ir-192 source. This is a more efficient method than TLD dosimetry at discrete and distant positions. Relative to TLD dosimetry, it is found to be better reproducible, easy to use and a less expensive method of dosimetry.

Atomic force microscopy investigation of the dependence of cellular elastic moduli on glutaraldehyde fixation.

Summary The atomic force microscope (AFM) has provided nanoscale analyses of surfaces of cells that exhibit strong adhesive and cell spreading properties. However, it is frequently reported that prior fixation is required for reliable imaging of cells with lower adhesive properties. In the present study, the AFM is used to assess the effects of fixation by glutaraldehyde on the elastic modulus of a human rhabdomyosarcoma transfectant cell line RDX2C2. Our results show a sharp increase in the elastic modulus for even mild fixation (0.5% glutaraldehyde for 60 s), accompanied by a dramatic improvement in imaging reproducibility. An even larger increase is seen in NIH-3T3 mouse fibroblasts, although in that case fixation is not typically necessary for successful imaging. In addition, our results suggest that treatment with glutaraldehyde restricts the content of the resulting images to features nearer to the cell surface.

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells.

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells.

Integrin alpha2beta1 modulates EGF stimulation of Rho GTPase-dependent morphological changes in adherent human rhabdomyosarcoma RD cells.

The ability of cells to undergo shape changes is essential for diverse cellular functions including cell growth, differentiation, and movement. The present study examines how an integration of the function of alpha2beta1 integrin with that of the receptor for epidermal growth factor (EGFR) modulates EGF-stimulated morphological changes in human rhabdomyosarcoma RD transfectant cells. Upon EGF stimulation, RD transfectant cells that lacked alpha2beta1 integrin expression (RDpF) underwent contraction; in contrast, expression of alpha2beta1 on RD cells (RDX2C2) resulted in transient cell spreading. Integrin alpha2 cytoplasmic domain played a critical role in the observed alpha2beta1-mediated conversion from a cell rounding to a cell spreading phenotype. Thus, the expression of an alpha2 cytoplasmic domain deletion variant (X2C0) or a chimeric alpha2beta1 containing the cytoplasmic domain of alpha4 (X2C4) or alpha5 (X2C5), instead of alpha2, failed to mediate spreading upon EGF stimulation. Using dominant negative (DN) mutants of RhoGTPases, results revealed that RhoA activation was required for both EGF-stimulated responses of cell rounding and spreading, Cdc42 functioned in the re-spreading of cells after undergoing EGF-stimulated contraction, and Rac1 was required in alpha2beta1-mediated RD cell spreading. Therefore, alpha2beta1 integrin function can switch the Rho GTPase-dependent cell shape changes in RD cells from an EGF-stimulated cell contraction to a spreading morphology. Together, results show that integrin alpha2 cytoplasmic domain plays an indispensable role in the ability of integrin alpha2beta1 to modulate EGF stimulation of Rho-GTPase-dependent morphological changes in RD cells.

beta1 Integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation.

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 microg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1beta > RANTES MIP-1alpha. In contrast, using filters coated with fibronectin (20 microg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic: thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 microg/mL). To determine the involvement of beta1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to alpha2beta1; however, complete inhibition required a combination of mAbs to alpha1beta1 and alpha2beta1. In comparison, migration on fibronectin was inhibited by mAb to alpha3beta1 and alpha5beta1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1alpha, MIP-1beta, and RANTES), as well as the nature and concentration of the ECM substrate involved.

Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides.

Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.

A differential role of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cell movement.

Rat pheochromocytoma PC12 cells have been widely used as a cell system for study of growth factor-stimulated cell functions. We report here that nerve growth factor (NGF) stimulated both chemotaxis (directional migration) and chemokinesis (random migration) of PC12 cells. Treatment with a MEK1/2-specific inhibitor (PD98059) or expression of a dominant negative variant of Ras differentially inhibited NGF-stimulated chemotaxis but not chemokinesis of PC12 cells. Priming of PC12 cells with NGF resulted in reduced extracellular signal-regulated kinase (ERK) activation and loss of chemotactic, but not chemokinetic, response. In addition, NGF stimulation of ERK is known to involve an early transient phase of activation followed by a late sustained phase of activation; in contrast, epidermal growth factor (EGF) elicits only early transient ERK activation. We observed that like NGF, EGF also stimulated both chemotaxis and chemokinesis, and treatment with PD98059 abolished the EGF-stimulated chemotaxis. Therefore, the early transient phase of ERK activation functioned in signaling chemotaxis; the late sustained phase of ERK activation did not seem to have an essential role. In addition, our results suggested that chemotactic signaling required a threshold level of ERK activation; at below threshold level of ERK activation, chemotaxis would not occur.

Characterization of a functional relationship between hepatocyte growth factor and mouse bone marrow-derived mast cells.

During the early stage (at 4 weeks) of interleukin-3 (IL-3)-induced development, mouse bone marrow-derived mast cells (BMMC) express alpha 4, alpha 5 and alpha 6 integrins, whereas with further maturation beyond 10 weeks, only alpha 5 integrin remains stably expressed. Hepatocyte growth factor (HGF) modulates the growth and movement of diverse cell types upon binding to its receptor, encoded by the proto-oncogene c-met. We report here the expression of c-met by BMMC throughout the course of their development. In addition, HGF stimulated migration of early week-4 BMMC, but not of the later stage week-10 BMMC, on fibronectin and laminin substrates. The developmental stage-dependent effect of HGF on BMMC was due to specific stimulation of the migratory function of alpha 4 and alpha 6, but not alpha 5 integrins. In addition, HGF had no effect on BMMC growth, either alone or in combination with IL-3. While HGF is stimulatory of the migratory function of BMMC, our results show that BMMC in turn can modulate HGF function. Thus, upon activation via the IgE receptors, BMMC released proteases that abolished HGF activities. Analyses of the degradation products by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot using antisera prepared against recombinant HGF and the kringle 3 domain of HGF revealed specific degradation of HGF alpha but not beta/beta' subunits. Therefore, our results suggest that: 1) the motogenic effect of HGF on BMMC varies according to the stage of their development, 2) HGF stimulation of BMMC migration is due to selective activation of alpha 4 and alpha 6, but not alpha 5 integrin function, and 3) there exists a two-way relationship between BMMC and HGF such that HGF stimulates the beta 1 integrin-mediated migratory function of BMMC, which can, in turn, modulate HGF function by release of serine proteases.

Leukocytes utilize both alpha4 and alpha5 integrins for intraislet infiltration in non-obese diabetic mice.

The loss of insulin-producing cells during the development of type 1 diabetes is dependent on leukocyte infiltration of beta-islets in the pancreas. Injection of antibodies to integrins and their ligands has been shown to prevent the development of diabetes in non-obese diabetic (NOD) mice. However, little is known about the progression of infiltration by leukocytes after their homing and extravasation into the pancreas. In the present study, the effect of monoclonal antibodies (mAbs) to alpha4 and alpha5 integrins on leukocytes that had infiltrated the islets was characterized in NOD mice at 10 weeks of age when insulitis was in progress, or in mice with recent onset of diabetes. Injection of mAbs to either alpha4 or alpha5 integrins had little effect on the extent of leukocyte infiltration in 10-week-old or diabetic mice. In contrast, leukocyte infiltration was significantly reduced upon injection of mAbs to both alpha4 and alpha5 integrins. The reduction in leukocyte infiltration was due to decreases in the percentage of islets with intraislet infiltration. However, the observed effect of mAbs to alpha4 and alpha5 integrins was reversible, since intraislet infiltration resumed upon termination of antibody treatment. Results suggest that after homing to the pancreas, leukocytes utilize both alpha4 and alpha5 integrins for intraislet infiltration.

Tolerance induction by acylated peptides: effect on encephalitogenic T cell lines.

We reported previously that acylation of an encephalitogenic peptide of myelin basic protein (MBP68-86) by attachment of palmitoyl chloride (PAL68-86) converted this peptide into a powerful tolerogen for EAE in the Lewis rat. In this study we show that T cell lines derived from a PAL68-86-protected rat proliferated poorly to MBP68-86 in vitro, even after repeated passages in this peptide and IL-2. Conversely, T cell lines derived from untreated rats that were challenged with MBP68-86 or PAL68-86 in CFA responded vigorously to MBP68-86 when propagated for many passages in this peptide but became gradually unresponsive after being propagated in the presence of PAL68-86. The modulation of the T cell lines by PAL68-86 in vitro was reflected by a significant reduction in their ability to transfer EAE to recipients. A high percentage of cells stained with an anti-Vbeta8.2 antibody, regardless of whether they were propagated in the presence of unmodified or acylated peptide. The results are consistent with the notion that tolerance induced by PAL68-86 operates by functional inactivation and provide the basis for the use of acylated peptides in the antigen-specific treatment of autoimmune diseases.

Treatment of cardiac allografts with established leukocyte infiltration by modulation of alpha4 (CD49d) and leukocyte function-associated antigen-1 (CD11a/CD18) integrin function.

BACKGROUND: Leukocyte infiltration is a landmark feature of organ rejection. The present study was undertaken to determine whether monoclonal antibodies (mAb) against alpha4 (CD49d) and/or leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) would reverse ongoing rejection in a mouse C57BL/6-to-BALB/c heart transplant model. METHODS: Control animals had rejection on postoperative day (POD) 8. Treatment with mAb started on POD 4 when leukocyte infiltration was well established. The recipients were treated with (1) mAb LFA-1, (2) mAb alpha4, and (3) mAbs LFA-1 + alpha4 at a dose of 6 mg/kg/day i.v. on PODs 4, 5, and 7. Untreated and rat IgG-treated animals were used as controls. RESULTS: Control animals experienced rejection on POD 8. Treatment with mAb against LFA-1 or alpha4 alone prolonged allograft survival to 17.0+/-3.2 and 24.3+/-4.6 days, respectively (P < 0.01 vs. controls). Combination therapy with both mAb increased allograft survival to 28.2+/-3.7 days (P < 0.01 vs. controls). Sequential pathological studies showed the mAb to alpha4, but not LFA-1, markedly reduced the degree of lymphocytic infiltration in cardiac allografts. In contrast, a different pattern was observed using in vitro studies: mAb to LFA-1, not alpha4, significantly reduced proliferative responses in mixed lymphocyte culture and interleukin-2 production from recipient splenocytes on POD 8. CONCLUSION: These data indicate that integrins play an important role in rejection. Although the effect of mAb against alpha4 and LFA-1 may involve different mechanisms, treatment with mAbs to integrins may be valuable in future clinical transplantation by averting ongoing rejection and prolonging graft survival.

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver.

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.

An epitope on VLA-6 (alpha6beta1) integrin involved in migration but not adhesion is required for extravasation of murine melanoma B16F1 cells in liver.

VLA-6 (alpha6beta1) integrin represents the major receptor for interaction with laminin substrate. It has been proposed that VLA-6 mediates tumor cell adhesion to the endothelium during extravasation. We have further explored this possibility using mouse melanoma B16F1 cells, which express VLA-6 as the principal laminin receptor, and two VLA-6 monoclonal antibodies (mAbs), MA6 and GoH3. Adhesion is a prerequisite of cell movement on matrix proteins. Thus, GoH3, which inhibited VLA-6-mediated adhesion, blocked cell movement on laminin. The recently prepared alpha6 integrin-specific mAb MA6 bound to an epitope in close proximity to GoH3, but it had no effect on VLA-6-mediated cell adhesion. We report here that although MA6 did not affect adhesion, it blocked mouse melanoma B16F1 cell movement on laminin to the same extent as GoH3. Results therefore demonstrate an active role of VLA-6 in providing cell movement as well as the initial adhesive event on laminin. In addition, mAb MA6 had no effect on the induction of tyrosine phosphorylation of focal adhesion kinase upon adhesion of B16F1 cells to laminin. Therefore, inhibition of cell movement by MA6 involved mechanism(s) other than an interference of VLA-6 signaling events leading to phosphorylation of focal adhesion kinase. The epitopes of GoH3 and MA6 may represent spatially and temporally related sites on VLA-6 that are involved during cell movement, or, alternatively, MA6 may inhibit the interaction of VLA-6 with associated cell surface molecules required for cell movement. In vivo videomicroscopy experiments also revealed that an inhibition of VLA-6 migratory function by MA6 resulted in a reduction in the ability of B16F1 to extravasate during hematogenous metastasis in the liver.

The role of integrins and extracellular matrix in anchorage-independent growth of a mammary carcinoma cell line.

Anchorage-independent growth is a property of malignant cells. Extracellular matrix proteins are present in tumor spheroids but their function is not clearly defined. In this paper we show that a murine mammary carcinoma cell line, SP1, which expresses the fibronectin receptor alpha 5 beta 1 requires fibronectin for anchorage-independent growth in soft agar. Growth factors (hepatocyte growth factor and transforming growth factor-beta) also promote SP1 colony growth. In contrast, collagen types I and IV have an inhibitory effect on SP1 colony growth. A clone isolated from SP1 cells which expresses the collagen/laminin receptor alpha 2 beta 1 as well as the fibronectin receptor alpha 5 beta 1, demonstrates increased colony formation in the presence of fibronectin and collagen. These data suggest a role for both the alpha 5 beta 1 and alpha 2 beta 1 integrin receptors in the regulation of anchorage-independent growth of mammary carcinoma cells.

Fibroblast growth factor-2 potentiates vascular smooth muscle cell migration to platelet-derived growth factor: upregulation of alpha2beta1 integrin and disassembly of actin filaments.

Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering beta1 integrin-mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of alpha2beta1, alpha3beta1, and alpha5beta1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding alpha2beta1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen-coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2-induced elongation and migration on collagen were inhibited by a blocking anti-alpha2beta1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle alpha-actin-containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of alpha2beta1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of alpha2beta1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.

Integrin VLA-6 (alpha 6 beta 1) mediates adhesion of mouse bone marrow-derived mast cells to laminin.

The development of mast cells from bone marrow precursors and their function as the mucosal- or connective-tissue-type mast cell are critically dependent on microenvironmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microenvironment. Recent studies have described multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow-derived mast cells (BMMC) and long-term mast cell lines to Engelbreth-Holm-Swarm (EHS) tumor laminin, rat laminin, human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA-alpha 6 integrin-specific mAb (MA6) together with the previously described mAb GoH3, VLA-6 (alpha 6 beta 1) integrin was found to be expressed and utilized by BMMC and long-term mast cell lines. VLA-6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67-kDa laminin receptor. Therefore, in addition to the 32/67-kDa laminin receptor described in early studies, BMMC also express VLA-6 integrin, which may have roles in the regulation of their development.

Differential utilization of VLA-4 (alpha 4 beta 1) and -5 (alpha 5 beta 1) integrins during the development of mouse bone marrow-derived mast cells.

Cytokines have been shown to have major roles in the development of mast cells from bone marrow progenitors. Immature mast cells derived from bone marrow thus leave the blood system to complete their course of maturation within tissues. However, it is now clear that VLA (beta 1) integrins with function in mediating cell-cell and cell-extracellular matrix protein interactions have effects on the growth and differentiation of diverse cell types. At present, the involvement of VLA integrins during mast cell development is still unclear. In this study, we report the preparation of a new monoclonal antibody (mAb) against mouse VLA-5 (alpha 5 beta 1) integrin. Together with mAb R1-2, we characterized the expression of VLA-4 (alpha 4 beta 1) and VLA-5 integrins, the two major fibronectin receptors, on two long-term cultured mast cell lines, CFTL-15 and MC/9. CFTL-15 cells were found to express both VLA-4 and -5 integrins whereas MC/9 cells expressed only VLA-5 but not VLA-4. We speculated that VLA integrin expression may be related to mast cell development. Thus bone marrow-derived mast cells (BMMC) were characterized after varying periods of development induced by IL-3. During the first 3 weeks the expression of VLA-4 and VLA-5 increased progressively and both were involved in mediating adhesion of BMMC to fibronectin. At time periods of greater than 3 weeks, the expression of VLA-4 declined gradually to little, if any, by week 13. In comparison, VLA-5 remained stably expressed and functioned as the major receptor for fibronectin. Results from this study therefore suggest that BMMC differentially utilize VLA-4 and VLA-5 integrins during IL-3-induced development. Differential expression of VLA integrins may have effects on the recirculation properties, tissue distribution and eventual maturation of progenitors to fully matured mast cells.