Rapid G protein-regulated activation event involved in lymphocyte binding to high endothelial venules.

The homing of blood borne lymphocytes into lymph nodes and Peyer's patches is mediated in part by recognition and binding to specialized high endothelial venules (HEV). Here we demonstrate that a rapid pertussis toxin-sensitive lymphocyte activation event can participate in lymphocyte recognition of HEV. In situ video microscopic analyses of lymphocyte interactions with HEV in exteriorized mouse Peyer's patches reveal that pertussis toxin has no effect on an initial "rolling" displayed by many lymphocytes, but inhibits an activation-dependent "sticking" event required for lymphocyte arrest. This is the first demonstration that physiologic lymphocyte-endothelial interactions can involve sequential rolling, activation, and activation-dependent arrest, previously shown only for neutrophils. The inhibitory effect of the toxin is dependent on its G protein-modifying ADP-ribosyltransferase activity and can be reversed by phorbol myristic acetate, which bypasses cell surface receptors to trigger activation-dependent adhesion. Lymphocyte sticking can occur within 1-3 s after initiation of rolling. We conclude that a rapid receptor-mediated activation event involving G protein signaling can trigger stable lymphocyte attachment to HEV in vivo, and may play a critical role in regulating lymphocyte homing.

Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.

Specific arrest of neutrophils in venules is central to their rapid accumulation during local inflammatory responses. Initial neutrophil rolling on endothelium is mediated by leukocyte L-selectin and the inducible vascular adhesion proteins P- and E-selectin. This rolling is a prerequisite for endothelial-dependent neutrophil arrest. Here we describe rolling of neutrophils on the surface of previously arrested neutrophils and demonstrate that this interaction involves L-selectin exclusively on rolling cells. The adherent neutrophil support of L-selectin-dependent neutrophil rolling in vivo can promote continuous and augmented leukocyte recruitment at sites of previous neutrophil accumulation.

High endothelial venule binding as a predictor of the dissemination of passaged murine lymphomas.

It has long been postulated that normal lymphocyte homing mechanisms help determine the metastatic spread of lymphoid neoplasms. The traffic of normal lymphocytes is controlled in part by the regulated expression of surface receptors for high endothelial venules (HEV), specialized venules that mediate the extravasation of circulating lymphocytes from the blood into lymphoid organs and sites of chronic inflammation. Here we have compared the in vivo growth patterns of HEV-binding vs. nonbinding murine lymphomas passaged intramuscularly into syngeneic recipients. We report that lymphomas that bind well to HEV (as assessed in a quantitative in vitro assay) disseminate widely via the blood, involving all lymph node groups symmetrically. Although both HEV-binding and nonbinding lymphomas gain access to the blood, gross involvement of lymph nodes by nonbinding lymphomas is limited to nodes draining local tumor at the site of injection, a prominent feature of these lymphomas; distant lymph nodes are not enlarged. The results suggest that the expression of functional receptors for HEV either controls the hematogenous dissemination of malignant lymphocyte populations to HEV-bearing organs, or is coregulated with factors determining this metastatic behavior. The findings support the concept that normal lymphocyte homing mechanisms are important to the spread of leukemias and lymphomas.

Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells.

The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Inflammation-induced endothelial cell adhesion to lymphocytes, neutrophils, and monocytes. Role of homing receptors and other adhesion molecules.

Adhesion to the vascular endothelium precedes or is a necessary prelude to leukocyte migration into the underlying tissue. Constitutive lymphocyte trafficking through lymphoid organs is controlled by tissue-specific interactions between molecules expressed on the surface of the lymphocyte (homing receptors) and ligands (vascular addressins) expressed on endothelial cells (HEV) within lymphoid tissues. Preliminary evidence suggests that lymphocytes may employ related but distinct interactions in their entry into some chronic sites of inflammation. Other leukocytes, such as neutrophils and monocytes, express molecules related or identical to lymphocyte homing receptors, and these molecules are exquisitely regulated by chemotactic factors and appear to be involved in the homing of these cells to inflamed tissues. In addition, inflammation in vivo induces increased endothelial cell adhesiveness for leukocytes that undoubtedly plays a key role in regulating leukocyte extravasation. Tissue- and inflammation-specific leukocyte/endothelial cell adhesion molecules constitute attractive targets for suppression or manipulation of the early stages of tissue inflammation.

"Allergic breakthrough" after antigen sensitization: height of IgE synthesis is temporally related to diurnal variation in endogenous steroid production.

Mice of the SJL strain normally display the low IgE responder phenotype. In the course of extensive studies on IgE responses with this strain of mice, it because apparent that the magnitudes of IgE antibody synthesis by SJL mice varied after antigen sensitization either alone or in conjunction with exposure to optimal enhancing doses of whole body irradiation. Analysis of the basis for this variability revealed that SJL respond to antigen sensitization by displaying cyclical IgE response curves determined by the time of day the sensitization is carried out. Moreover, when these low responders are converted to the high responder phenotype by low dose irradiation, the time of irradiation determines the magnitude of the IgE response obtained, whereas the time of antigen challenge determines the potential for generating a good response. The diurnal curve of IgE responses in both unirradiated and irradiation-enhanced low responder mice appears to be determined by normal variations in levels of endogenous corticosteroids, as indicated by the correlation between the normal diurnal curve of endogenous steroid production with the cyclical pattern of IgE responses. This apparent effect of endogenous steroid production on IgE antibody synthesis is supported by experiments demonstrating that the normal IgE pattern in both unirradiated and irradiated mice can be perturbed by administration of cortisone at the proper time of day. These results indicate that IgE antibody synthesis displays unique sensitivity to fluctuations in plasma steroid levels and may have significant implications for the role of steroids in the pathogenesis and management of allergic diseases.

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes.

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas.

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct roles of L-selectin and integrins alpha 4 beta 7 and LFA-1 in lymphocyte homing to Peyer's patch-HEV in situ: the multistep model confirmed and refined.

Circulating lymphocytes home to the mucosal lymphoid organs, Peyer's patches (PP), through high endothelial venules (HEV). In situ analyses revealed that transfused lymph node cells (LNCs) interact with PP-HEV in a series of overlapping adhesion events: L-selectin (CD62L) > alpha 4 beta 7 initiates interaction, L-selectin and alpha 4 beta 7 both participate in rolling, and G alpha i-linked activation triggers arrest that requires both alpha 4 beta 7 and LFA-1. alpha 4 beta 7 dramatically reduces rolling velocity, and appears to be required for engagement of LFA-1. In contrast with resting LNC, preactivated LNC or alpha 4 beta 7hi lymphoma cells require only alpha 4 beta 7 for arrest in PP-HEV. The predominant PP-HEV ligand for alpha 4 beta 7 but also apparently for L-selectin is the mucosal addressin MAd-CAM-1. These results validate the concept of multimolecular adhesion/decision cascades in physiologic lymphocyte-endothelial recognition, define a novel role for alpha 4 integrins as a "bridge" between selectin and beta 2 integrin-dependent events, and reemphasize the potential for direct adhesion through preactivated alpha 4 integrins alone.

Induction of immunologic tolerance to the trimellitate haptenic group in mice: model for a therapeutic approach to trimellitic anhydride-induced hypersensitivity syndromes in humans?

IgE and IgG antibody responses to the trimellitate (TM) hapten group were elicited in mice by the intraperitoneal injection of the hapten coupled with keyhole limpet hemocyanin (KLH). Treatment of such previously sensitized mice with the same hapten coupled to synthetic copolymer of D-glutamic acid and D-lysine (D-GL) after primary immunization resulted in significant and specific suppression of anti-TM antibody responses in both IgE and IgG classes. These results provide direct evidence for the potential clinical usefulness of the D-GL immunotherapeutic approach to TMA sensitivity in humans.

Human lymphocyte and lymphoma homing receptors.

The migration of normal and malignant lymphocytes is controlled in part by selective lymphocyte recognition of high endothelial venules (HEV) at sites of lymphocyte exit from the blood. Recirculating lymphocytes appear to utilize structurally related, yet functionally distinct, 90-kD receptors to interact in an organ-specific manner with HEV in peripheral lymph nodes, in mucosa-associated lymphoid tissues (Peyer's patches, appendix), and in inflamed joint tissue (synovium). These lymphocyte "homing receptors" constitute a family of glycoprotein endothelial cell recognition elements that regulate the extravasation of circulating normal and neoplastic lymphocytes into different organs of the body, and thus play an important role in determining the characteristics of local immune responses and the patterns of dissemination of lymphoid neoplasms.

Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies.

Endothelial cells play an essential role in immune responses by regulating the entry of leukocytes into lymphoid tissues and sites of inflammation. As an initial approach to analyzing endothelial cell specialization in relation to such immune function, we have produced monoclonal antibodies (MAB) against mouse lymph node endothelium. Three antibodies were selected: MECA-20, recognizing the endothelium of all blood vessels in lymphoid as well as non-lymphoid organs; MECA-217, which stains the endothelium lining large elastic arteries, but among small vessels is specific for post-capillary venules within lymphoid organs and tissues exposed to exogenous antigen, such as skin and uterus; and MECA-325, an antibody that demonstrates specificity for the specialized high endothelial venules (HEV) that control lymphocyte homing into lymph nodes and Peyer's patches. MECA-325 failed to stain vessels in any non-lymphoid organs tested. Immunoperoxidase studies of HEV in lymph node frozen sections, and of isolated high endothelial cells in suspensions, demonstrated that the antigens recognized by all three antibodies are expressed at the cell surface; those defined by MECA-20 and MECA-325 are also present in the cytoplasm. To study the regulation of the antigens defined by these MAB in relation to extra-lymphoid immune reactions, we assessed their expression in induced s.c. granulomas as a model for chronic inflammation. Small vessels in the granulomas were already stained by MECA-217 in the first days of development. In contrast MECA-325 detected postcapillary venules (which frequently displayed the morphologic characteristics of HEV) only from approximately 1 wk, in parallel with the development of a persistent mononuclear cell infiltrate including numerous lymphocytes. The selective appearance of the MECA-325 antigen on vascular endothelium supporting lymphocyte traffic in both lymphoid and extra-lymphoid sites suggests that this antigen may play an important role in the process of lymphocyte extravasation. The demonstration of lymphoid organ- and inflammation-specific microvascular antigens offers direct evidence for a complex specialization of endothelium in relation to immune stimuli, and supports the concept that microvascular differentiation may play an important role in local immune responses.

Regulation of IgE antibody production by serum molecules. V. Evidence that coincidental sensitization and imbalance in the normal damping mechanism results in "allergic breakthrough".

Experiments presented in this paper were designed to test a new concept concerning the possible pathogenesis of the allergic phenotype. This concept, termed "allergic breakthrough" considers that one of the avenues toward the allergic phenotype involves coincidental sensitization combined with an imbalance in the normal damping mechanism that serves to limit IgE antibody production. The three predictions of this concept that can be tested experimentally are: 1) manipulations that are effective in heightening or re-establishing the damping mechanism should manifest persistence insofar as IgE antibody synthesis to the relevant allergen is concerned; 2) once allergic breakthrough has occurred, the height of production of IgE antibodies specific for the sensitizing agent should remain elevated at levels characteristic of the allergic phenotype, even after the threshold of damping activity has returned to a normal level; and 3) allergic breakthrough should display specificity in that breakthrough would occur in response to subsequent exposure to the specific antigen to which coincidental sensitization initially occurred, but not for other unrelated antigens. The studies presented herein confirm each one of these predictions, thereby providing substantial support for the validity of this concept as one possible distinguishing feature between individuals manifesting the nonallergic and allergic phenotypes, respectively.

Regulation of IgE antibody production by serum molecules. IV. Complete Freund's adjuvant induces both enhancing and suppressive activities detectable in the serum of low and high responder mice.

Two biologically active serum molecules manifesting precisely opposite biologic effects, both of which are selective for IgE antibody synthesis, can be detected in the serum and ascites fluids of CFA-immune mice. One activity, described previously, is suppressive and hence termed suppressive factor of allergy (SFA); the other, reported for the first time herein, is enhancing and has been termed enhancing factor of allergy (EFA). The ability to detect one vs the other activity requires certain special manipulations such as different doses of low dose x-irradiation. Conclusive evidence for the existence of two distinct factors mediating these two opposing biologic effects was obtained in studies demonstrating that affinity chromatography on concanavalin A-Sepharose segregated the two molecular entities. Thus, SFA binds poorly or not at all to Con A-Sepharose, whereas EFA binds to Con A and can be recovered in the eluate eluted with the competitive sugar alpha-methyl-D-glucopyranoside.

L-selectin-mediated lymphocyte rolling on MAdCAM-1.

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.

A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man.

We describe a 90-kDa lymphocyte surface glycoprotein, recognized by the monoclonal antibody Hermes-1, that is involved in endothelial cell recognition and lymphocyte trafficking in man. This molecule is selectively expressed on normal or transformed lymphoid cells that are able to recognize and bind to high endothelial venules (HEV, specialized vessels that mediate lymphocyte exit from the blood into lymphoid organs); appears to be linked to HEV recognition function since, in fluorescence-activated cell sorting of variants of a cloned cell line, HEV binding ability co-selects with the Hermes-1 antigen; bears the predominant cell surface epitopes recognized by heterologous anti-human lymphocyte antibodies able to interfere with lymphocyte binding to HEV; and is structurally similar to a previously described mouse lymphocyte surface receptor for HEV. These findings demonstrate that the molecule defined by Hermes-1 either functions as a specific lymphocyte surface receptor for HEV, or is both precisely coregulated and physically and/or functionally associated with such receptors. The expression of this putative receptor for HEV on normal human lymphocyte populations parallels, and thus presumably helps determine, their migratory status in vivo. Hermes-1 should be a powerful tool for analyzing the role of endothelial cell recognition in the traffic of normal and neoplastic human lymphocytes.

Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion.

P-selectin, an inducible cell adhesion molecule, mediates rolling of neutrophils on activated vascular endothelium. Because rolling is an early event of the inflammatory response, therapeutic applications of selectin antagonists have been of broad interest. There are, however, no truly satisfactory therapeutic candidates among known inhibitors. Consequently, we have used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, a process based on oligonucleotide combinatorial chemistry and in vitro selection, to develop aptamer antagonists of P-selectin. Equilibrium dissociation constants for aptamer/P-selectin binding range from 16 to 710 pM, a 10(5)-10(6)-fold improvement compared with the minimal carbohydrate ligand, sialyl Lewis X (sLeX). Aptamer binding is divalent cation dependent and, unlike sLeX, is specific for P-selectin. The selectivity for human P-selectin relative to human E-selectin or human L-selectin is 10(4)-10(5). In vitro, aptamers bind with subnanomolar affinities to P-selectin expressed on thrombin-activated platelets, inhibit the binding of P-selectin-IgG chimera to sLeX and to neutrophils, and block the binding activated platelets to neutrophils in flow cytometry and in hydrodynamic assays. Extrapolating from their in vitro characteristics, these novel P-selectin-specific antagonists may be suitable candidates for therapeutic development.

A tissue-specific endothelial cell molecule involved in lymphocyte homing.

An endothelial cell surface molecule that is selectively expressed in mucosal organs is required for lymphocyte homing to mucosal lymphoid tissues. This 'vascular addressin' appears to function as a tissue-specific marker or address signal for recognition by lymphocytes circulating in the blood.

Immunologic tolerance to allergenic protein determinants: properties of tolerance induced in mice treated with conjugates of protein and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL).

Conjugates of proteins and the synthetic copolymer of D-glutamic acid and D-lysine (protein-D-GL) reproducibly induce significant unresponsiveness to the protein antigens in experimental mice. Proteins studied include ovalbumin and antigen E of ragweed extract, the major allergen of ragweed pollen. The unresponsive state 1) can be induced in both unsensitized and previously sensitized experimental animals, 2) is selectively confined to responses of the IgE antibody class, 3) is long lasting, and 4) is highly antigen specific. IgE antibody responses can also be suppressed by administering comparable doses of unconjugated protein alone; however, the unresponsive state induced in this manner is only transient and rebound production of IgE antibody is often observed. Results from the studies of the cellular basis of the protein-D-GL induced unresponsiveness demonstrate that 1) protein-D-GL conjugates do not induce unresponsiveness at the level of protein-specific B cells, 2) tolerance is not induced by virtue of a detectable antigen-specific suppressor T cell mechanism, 3) tolerance is most probably induced in the antigen-specific helper T cell populations. The significant IgE-selective and antigen-specific tolerogenic activity of protein-D-GL conjugates make these compounds potential candidates for use as therapeutic agents in the treatment of IgE-mediated human allergic disorders induced by protein allergens.