Charles A. West

Microangiectasias: Structural regulators of lymphocyte transmigration

The migration of lymphocytes into inflammatory tissue requires the migrating cell to overcome mechanical forces produced by blood flow. A generally accepted hypothesis is that these forces are overcome by a multistep sequence of adhesive interactions between lymphocytes and endothelial cells. This hypothesis has been recently challenged by results demonstrating wall shear stress on the order of 20 dyn/cm2 in vivo and infrequent lymphocyte–endothelial adhesion at wall shear stress >1–2 dyn/cm2 in vitro. Here, we show that lymphocyte slowing and transmigration in the skin is associated with microangiectasias, i.e., focal structural dilatations of microvessel segments. Microangiectasias are inducible within 4 days of the onset of inflammation and lead to a greater than 10-fold local reduction in wall shear stress. These findings support the hypothesis that a preparatory step to lymphocyte transmigration involves structural adaptations in the inflammatory microcirculation.

Stochastic Regulation of Cell Migration from the Efferent Lymph to Oxazolone-Stimulated Skin

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks ∼96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.

Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

Aldehyde Fixation of Thiol-reactive Fluorescent Cytoplasmic Probes for Tracking Cell Migration

Tracking of cell migration plays an important role in the study of morphogenesis, inflammation, and metastasis. The recent development of probes that exist as intracellular peptide-fluorescence dye adducts has offered the possibility of aldehyde fixation of these dyes for detailed anatomic studies of lymphocyte trafficking. To define the conditions for fixation of these cytoplasmic fluorescent probes, we compared fixation conditions containing formaldehyde, glutaraldehyde, paraformaldehyde, zinc formaldehyde, and glyoxylate, as well as fixation by quick-freezing in liquid nitrogen-cooled methylbutane. The efficacy of aldehyde fixation of the cell fluorescence was assessed by quantitative tissue cytometry and flow cytometry. We studied cytoplasmic fluorescent dyes with discrete emissions in the green [5-chloromethylfluorescein diacetate (CMFDA); 492 ex, 516 em] and orange [5-(and-6)-(4-chloromethyl(benzoyl)amino) tetramethylrhodamine (CMTMR); 540 ex, 566 em] spectra. The results demonstrated that aldehyde fixation preserved cell fluorescence for more than 6 months. The primary difference between the aldehyde fixatives was variability in the difference between the yield of the cell fluorescence and the relevant background fluorescence. Formaldehyde and paraformaldehyde were superior to the other fixatives in preserving cell fluorescence while limiting background fluorescence. With these fixatives, both the CMFDA and CMTMR fluorescent dyes permitted sufficient anatomic resolution for reliable localization in long-term cell tracking studies.

EFFECT OF SHEAR STRESS ON EFFERENT LYMPH-DERIVED LYMPHOCYTES IN CONTACT WITH ACTIVATED ENDOTHELIAL MONOLAYERS

SummaryLymphocyte interactions with endothelial cells in microcirculation are an important regulatory step in the delivery of lymphocytes to peripheral sites of inflammation. In normal circumstances, the predicted wall shear stress in small venules range from 10 to 100 dyn/cm2. Attempts to measure the adhesion of lymphocytes under physiologic conditions have produced variable results, suggesting the importance of studying biologically relevant migratory lymphocytes. To quantify the effect of shear stress on these migratory lymphocytes, we used lymphocytes obtained from sheep efferent lymph ducts, defined as migratory cells, to perfuse sheep endothelial monolayers under conditions of flow. Quantitative cytomorphometry was used to distinguish cells in contact with the endothelial monolayers from cells in the flow stream. As expected, migratory cells in contact with the normal endothelial monolayer demonstrated flow velocities less than the velocity of cells in the adjacent flow stream. The flow velocities of these efferent lymphocytes were independent of cell size. To model the inflammatory microcirculation, lymphocytes were perfused over sequential endothelial monolayers to directly compare the velocity of cells in contact with cytokine-activated and unactivated control monolayers. The tumor necrosis factor and interleukin-1-activated endothelial monolayers marginally decreased cell velocities at 1.2 dyn/cm2 (3.6%), but significantly reduced cell velocities 0.3 dyn/cm2 (27.4%; P<0.05). Similarly, the fraction of statically adherent lymphocytes decreased as shear stress increased to 1.2 dyn/cm2. These results suggest that typical wall shear stress in small venules, of the order of 20 dyn/cm2, are too high to permit adhesion and transmigration of migratory lymphocytes. Additional mechanisms must be present in vivo to facilitate lymphocyte transmigration in the inflammatory microcirculation.

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction

Background. Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. Methods. In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. Results. The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. Conclusions. These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.

Dynamic deformation of migratory efferent lymph-derived cells “trapped” in the inflammatory microcirculation

The cellular immune response depends on the delivery of lymphocytes from the lymph node to the peripheral site of antigenic challenge. During their passage through the inflammatory microcirculaton, the migratory cells can become transiently immobilized or “trapped” in small caliber vessels. In this report, we used intravital microscopy and temporal area mapping to define the dynamic deformation of efferent lymph‐derived mononuclear cells trapped in the systemic inflammatory microcirculation. Mononuclear cells obtained from the efferent lymph draining the oxazolone‐stimulated microcirculation were labeled with fluorescent dye and reinjected into the feeding arterial circulation. Intravital video microscopy observed thousands of cells passing through the microcirculation; 35 cells were “trapped” in the oxazolone‐stimulated microcirculation. Temporal area maps of the trapped cells demonstrated dramatic slowing and deformation. The cells were trapped in the microcirculation for a median of 8.90 sec (range 5–17 sec) prior to returning to the flow stream. During this period, the cells showed sustained movement associated with both antegrade locomotion (mean cell velocity = 7.92 μm/sec; range 1.16–14.23 μm/sec) and dynamic elongation (median cell length = 73.8 μm; range 58–144 μm). In contrast, efferent lymph‐derived cells passing unimpeded through the microcirculation demonstrated rapid velocity (median velocity = 216 μm/sec) and spherical geometry (median diameter = 14.6 μm). Further, the membrane surface area of the “trapped” cells, calculated based on digital image morphometry and corrosion cast scanning electron microscopy, suggested that the fractional excess membrane of the cells in the efferent lymph was significantly greater than previous estimates of membrane excess. These data indicate that transient immobilization of efferent lymph‐derived mononuclear cells in the systemic inflammatory microcirculation is rare. When “trapping” does occur, the shape changes and sustained cell movement facilitated by excess cell membrane may contribute to the return of the “trapped cells” into the flow stream.

Spatial variation of plasma flow in the oxazolone-stimulated microcirculation.

Abstract. Introduction: In cutaneous lymphocytic inflammation, enhanced regional blood flow is suggested by persistent erythema and warmth. Direct assessment of the microcirculation, however, has been limited by tissue edema and skin thickness.¶Methods: To assess the microcirculatory adaptations to the epicutaneous antigen oxazolone, we studied the first pass kinetics and microvascular topography of the inflammatory skin microcirculation using a specially adapted epi-illumination intravital microscopy system. The fluorescence intravital videomicroscopy and streaming image acquisition of fluorescein-labeled dextran (∼500,000 MW) injections were used to assess changes in plasma flow.¶Results: Direct plasma tracer injections of both the oxazolone-stimulated and control microcirculation demonstrated comparable transit times (leading edge and intensity-weighted peak times) from the carotid artery to the superficial vascular plexus (p>0.05). In contrast to transit times, continuous infusion of the plasma tracer demonstrated a significant increase in the delivery of the fluorescein-labeled dextran to the oxazolone-stimulated microcirculation. Quantitative morphometry of intravital microscopic images demonstrated a 2.2-fold increase in the mean diameter of vessels in the superficial vascular plexus (p<0.01). Further, fluorescence intensity mapping indicated that the increase was associated with increased perfusion of focal regions of the superficial vascular plexus (p<0.001).¶Conclusions: These results indicate that the oxazolone-stimulated adaptations of the inflammatory microcirculation include both microvascular dilatation and the redistribution of plasma flow.

Cell adhesion molecule expression in the sheep thymus

Cell adhesion molecules are potential regulating factors in both prethymic and intrathymic T cell development. An experimental challenge has been the development of a large animal model that facilitates in vivo studies of both intrathymic development and lymphocyte migration. To extend earlier studies of thymic development, we have developed a panel of monoclonal antibodies (mAb) to a variety of sheep cell adhesion molecules. Immunohistochemistry was used to define mAb reactivity and flow cytometry was used to quantify expression of cell adhesion molecules within the thymus. To facilitate flow cytometry definition of cortical thymocytes, mAbs were developed to the sheep CD1 antigen. Dual parameter flow cytometry provided a phenotypic characterization of cell adhesion molecule expression on both CD1(+) and CD1(-) sheep thymocyte populations. These studies demonstrated significantly enhanced cortical thymocyte expression of three cell adhesion molecules: beta1 integrin (CD29), ICAM-2 and LFA-3. The beta1 integrin cell adhesion molecule was also expressed at higher levels on CD1(+) thymocytes in post-natal lambs as compared to adult sheep. These studies of thymocyte membrane molecule expression should facilitate future investigations of sheep intrathymic development and T lymphocyte immigration.

Generation of Sheep X (Sheep X Mouse) Heterohybridoma Cell Line Expressing the Beta-1 Integrin Membrane Molecule

Sheep are an important biological model in such diverse areas as immunology and reproductive biology. The limitation of sheep as an experimental model is the absence of reliable cell lines. To establish cell lines that express functional sheep membrane molecules, we produced a sheep x mouse heterohybridoma by fusion of sheep efferent lymph T cells with the murine myeloma cell line NS1. A cloned heterohybridoma fusion partner was selected by treatment with 8-azaguanine. The resulting cell line HL1/385 was selected for hypoxanthine/aminopterin/thymidine (HAT) sensitivity and growth efficiency. The HL1/385 cell line was used as a back-fusion partner into lectin-stimulated efferent T lymphocytes. The back-fusion approach produced more than 50 heterohybrid cell lines with high growth efficiency. The expression of physiological levels of the sheep beta-1 integrin cell surface molecule on the HT4/6 cell line was stable for months in culture. These results suggest that somatic heterohybrids may provide a reliable source of cell lines for sheep studies in vitro.