Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules.

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions.

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L-selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L-selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo-glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.

Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow.

We demonstrate an additional step and a positive feedback loop in leukocyte accumulation on inflamed endothelium. Leukocytes in shear flow bind to adherent leukocytes through L-selectin/ligand interactions and subsequently bind downstream and roll on inflamed endothelium, purified E-selectin, P-selectin, L-selectin, VCAM-1, or peripheral node addressin. Thus adherent leukocytes nucleate formation of strings of rolling cells and synergistically enhance leukocyte accumulation. Neutrophils, monocytes, and activated T cell lines, but not peripheral blood T lymphocytes, tether to each other through L-selectin. L-selectin is not involved in direct binding to either E- or P-selectin and is not a major counterreceptor of endothelial selectins. Leukocyte-leukocyte tethers are more tolerant to high shear than direct tethers to endothelial selectins and, like other L-selectin-mediated interactions, require a shear threshold. Synergism between leukocyte-leukocyte and leukocyte-endothelial interactions introduces novel regulatory mechanisms in recruitment of leukocytes in inflammation.

CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

Low doses of interferon alpha result in more effective clinical natural killer cell activation.

To define critical parameters concerning interferon (IFN) effects upon natural killer (NK) cells in vivo, we gave cancer patients serial weekly intramuscular injections of purified lymphoblastoid IFN in six doses ranging from 10(5) to 3 X 10(7) U. Dose sequences were determined by randomly allocating patients to one of six levels in a latin square ordering scheme. NK cell stimulation, a threefold peak increase above preinjection levels of cytolysis (P = 0.022), occurred in peripheral mononuclear cells (PMC) sampled 24 h postinjection, of 3 X 10(6) U, but was not detectable at any dose in PMC sampled 7 d postinjection. No blunting occurred in NK cell responsiveness to repeated injection of IFN dosages a second time at or several weeks after study completion. At IFN doses of 3 X 10(6), 10(7), and 3 X 10(7) U, a negative correlation existed between the amount of IFN injected and the average extent of NK cell activation (r = -0.423, P less than 0.05). This contrasted with the progressively increasing response of NK cells to in vitro incubation with increasing concentration of up to 3,000 U/ml of IFN. Overnight culturing of PMC sampled before IFN injections resulted in a mean 1.9-fold increase in cytolytic activity (P = 0.0005) and a mean 53% decrease in variance (P = 0.024) between serial preinjection NK cell activity determinations. Cell separation procedures may, therefore, have resulted in NK cell inactivation, from which overnight culturing permitted recovery. We found that maximal NK cell activation at a low IFN dose, decreasing NK cell responsiveness at higher doses, and the need to culture PMC to efficiently detect NK cell boosting may account for disparities in reported effects of IFN on NK cell function.

Separation of human natural killer cell subpopulations differentially responsive to interferon potentiation.

Subsets of human natural killer (NK) cells were identified that differed in the capacity to be activated by interferon (IFN) or the IFN-inducer, polyriboinosinic:polyribocytidylic acid [poly(I):poly(C)]. These subsets, which represented effectors of both spontaneous and antibody-dependent cellular cytotoxicity, were physically separable on the basis of cell buoyant density changes induced by exposure of lymphocytes to hyperosmolar Ficoll-Hypaque solutions or by centrifugation of lymphocytes through hyperosmolar (350 mOs/kg) Percoll gradients. Hyperosmolar conditions per se altered neither cell viability, NK cell cytolytic activity, nor the capacity of NK cells in unseparated lymphocyte preparations to be activated by IFN. IFN-unresponsive NK cells, separated by centrifugation through a 350 mOs/kg Percoll layer of 1.069-1.070 g/cm3 specific density, constituted 20 +/- 4% of all active NK cells identified at the single cell level and, per active NK cell, killed comparably to unstimulated IFN-responsive NK cells in 51Cr release assays. Thus, the IFN-unresponsive phenotype was probably not attributable to NK cells that were in an activated state prior to IFN treatment. Surface marker analysis of active NK cells at the single cell level identified comparable proportions in each subfraction to be of the OKM1+, OKT8+, or OKT11+ phenotypes and few, if any, in either subfraction to be of the OKT3+ phenotype. The human IFN-unresponsive NK cell phenotype, in contrast to the corresponding phenotype in the mouse, was therefore not linked to expression of T-cell-associated membrane differentiation antigens.

Effects of interferon alpha on the sheep erythrocyte "receptor" of human lymphocytes.

The percentage of peripheral blood lymphocytes (PBLs) which formed rosettes with sheep erythrocytes declined from 62 +/- 2% to 29 +/- 4% (p = 0.001) when PBLs were incubated 18 h at 37 degrees C. In the presence of alpha interferon (IFN-alpha), a dose-dependent increase occurred in the percentage of sheep erythrocyte-binding PBL at the end of incubation compared with PBL incubated without IFN-alpha. Change in the number of sheep erythrocyte "receptors" (SER) probably did not account for the observed modulation of rosetting capacity, since the frequency and density of an SER-associated determinant (T11, as defined by immunofluorescence flow cytometry using the monoclonal antibody OKT11A) was unaffected by incubation with or without IFN-alpha. Treatment of PBL, control or IFN-alpha-treated, with neuraminidase (0.4 u/ml), restored rosetting capacity to levels characteristic of freshly prepared PBL. Neuraminidase did not affect rosetting or T11 expression by freshly prepared PBL, nor did it affect T11 expression on PBL cultured with or without IFN-alpha. We thus postulated that steric interference with SER function by sialic acid residues might result from de novo protein synthesis and glycosylation at the cell surface. Inhibition of either protein glycosylation by tunicamycin or protein synthesis by cycloheximide prevented the incubation-induced depression of rosetting capacity. IFN-alpha may modulate functional expression of SER and other surface receptors by altering cell-surface glycoprotein composition and distribution.

A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells.

We previously have obtained operational evidence of a hematopoietic cell L-selectin ligand expressed on normal human hematopoietic cells and on leukemic blasts. Using a technique developed in our laboratory for analyzing and identifying adhesion molecules, we show here that hematopoietic cell L-selectin ligand is a specialized glycoform of CD44. This L-selectin ligand activity of CD44 requires sialofucosylated N-linked glycans and is sulfation-independent. These data provide important insights on the structural biology of CD44 and reveal a role for this protein as an L-selectin ligand on human hematopoietic cells.

Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.

Studies of interleukin 1 (IL-1) alpha and beta have emphasized their functional similarities. IL-1 alpha and -beta are encoded by ancestrally related genes that have diverged dramatically in primary sequence; however, only modest differences in the regulation or biological activity of IL-1 alpha and IL-1 beta have been documented. Here we show that mouse L cells transfected with murine pro-IL-1 alpha cDNA expressed biologically active, 33-kilodalton pro-IL-1 alpha, and that this pro molecule was neither processed to the 17-kilodalton mature form nor secreted. The transfected cells also expressed membrane-associated IL-1 biological activity, indicating that the pro-IL-1 alpha cDNA can direct expression of membrane-associated IL-1 and that cleavage of the pro molecule is not required for membrane presentation. In contrast, transfection of pro-IL-1 beta cDNA did not generate biologically active material in L cells. Evidence is presented that the native murine IL-1 beta precursor molecule is also biologically inactive in peritoneal exudate cells stimulated with lipopolysaccharide. These differences in distribution of the bioactive forms of IL-1 alpha and IL-1 beta may provide selective advantages for the maintenance of two gene products with similar functions.

Regulation of antigen presentation. II. Anti-Ig and IL-2 induce IL-1 production by murine splenic B cells.

Murine splenic B cells did not constitutively express IL-1 activity. After culture with anti-Ig and T cell-conditioned media and then fixation, B cells expressed membrane IL-1 and were able to stimulate growth of the IL-1-dependent T cell clone D10. Expression of membrane IL-1 required stimulation of B cells for 2 days before fixation. Significant IL-1 activity was detectable in freeze-thaw lysates of identical B cell preparations by 12 h. B cells also released IL-1 into the culture media. In situ hybridization studies by using probes to murine IL-1 alpha and IL-1 beta genes supported these observations. Thus, messenger RNA for IL-1 alpha and IL-1 beta rose in parallel, were detected between 6 and 24 h of culture, and declined to low levels by 30 h. Despite the presence of mRNA for IL-1 alpha and IL-1 beta, only IL-1 alpha had functional activity as determined by the use of a mAb to IL-1 alpha. IL-2 was found to be an essential component of the T cell-derived supernatant. Although IL-4 or TNF did not induce significant B cell IL-1 expression, they both caused a modest, but reproducible enhancement when added in combination with IL-2. IFN-gamma, by contrast, partially inhibited IL-1 induction.

Interleukin-1 alpha and beta genes: linkage on chromosome 2 in the mouse.

Two interleukin-1 polypeptides, alpha and beta, are known, and cDNAs corresponding to each have been described. Genomic cloning and Southern blotting experiments suggest that in the mouse each is encoded by a gene present in one copy per haploid genome. Analysis of a panel of somatic cell hybrids carrying various mouse chromosomes on a constant Chinese hamster background indicates that both genes map to mouse chromosome 2. Further, analysis of the inheritance of DNA restriction fragment length polymorphisms associated with each gene in recombinant inbred strains of mice shows the two loci to be tightly linked to one another, and to lie approximately 4.7 centimorgans distal to B2m (beta-2 microglobulin). We have named the locus encoding IL-1 alpha Il-1 alpha and the locus encoding IL-1 beta Il-1b.

Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide.

Murine peritoneal exudate cells (PEC), analyzed immediately after isolation, did not express detectable IL 1 activity or IL 1-specific mRNA. Stimulation of these cells by adherence induced the expression of intracellular, membrane, and extracellular IL 1 activities within 4 hr. Analysis of mRNA from these cells showed a concurrent induction of both IL 1 alpha and IL 1 beta mRNA within 1 hr. However, this stimulation of IL 1 expression was transient, since PEC cultured for 5 days no longer expressed IL 1 bioactivity or specific mRNA. Stimulation of these quiescent cells with bacterial lipopolysaccharide induced the re-expression of intracellular, membrane, and extracellular IL 1 activities as well as IL 1 alpha and IL 1 beta mRNA. We found no qualitative difference in the degree or rate of induction of IL 1 alpha compared with IL 1 beta mRNA. These results indicate that resting macrophages are IL 1 negative, and that the IL 1 inducing stimuli used in this study act transiently to increase the levels of IL 1 alpha and IL 1 beta mRNA.

Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions.

Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewisa (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca(2+)-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin-expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions.

Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells.

T cells play a pathogenic role in many inflammatory and certain malignant skin diseases, including psoriasis, atopic and allergic contact dermatitis, and cutaneous T-cell lymphoma. Memory T cells that infiltrate the skin express a unique skin-homing receptor called cutaneous lymphocyte-associated antigen (CLA), a carbohydrate epitope that facilitates the targeting of T cells to inflamed skin. CLA is defined by both its reactivity with a unique monoclonal antibody, HECA-452, and its activity as a ligand for E-selectin, but the structure of the protein component of CLA has not previously been defined. Here we report that CLA is an inducible carbohydrate modification of P-selectin glycoprotein ligand-1 (PSGL-1), a known surface glycoprotein that is expressed constitutively on all human peripheral-blood T cells. Cultured peripheral-blood T cells can be differentiated into CLA-bearing cells, which bind both E-selectin and P-selectin, or CLA-negative cells, which bind P-selectin but do not bind E-selectin, suggesting that there is independent regulation of selectin-binding phenotypes. We propose that differential post-translational modification of a single cell-surface receptor, PSGL-1, mediated by fucosyltransferase VII, serves as a mechanism for regulating tissue-specific homing of memory T cells.

P-selectin, L-selectin, and alpha 4 integrin have distinct roles in eosinophil tethering and arrest on vascular endothelial cells under physiological flow conditions.

The adhesive interactions of eosinophils with purified E-, P-, and L-selectins; vascular cell adhesion molecule-1 molecule; and HUVEC were examined in shear flow. Compared with neutrophils, eosinophils showed markedly less binding to E-selectin, but significantly stronger avidity for P-selectin. Both cell types showed a similar level of tethering and rolling on L-selectin. Eosinophils tethered and arrested abruptly on vascular cell adhesion molecule-1. However, some of the tethers were detached within several seconds; this was prevented by stimulation with eotaxin. Eosinophils also showed immediate arrest on HUVEC stimulated with 100 U/ml TNF-alpha for 6 h. Treatment with L-selectin mAb decreased eosinophil accumulation on the HUVEC by abrogating secondary tethers through interactions between flowing and attached eosinophils. mAb to P-selectin but not to E-selectin strongly inhibited primary tethers and accumulation of eosinophils. mAb to the integrin alpha 4 subunit inhibited arrest, induced rolling or detachment of tethered eosinophils, and resulted in partial reduction of eosinophil accumulation. mAb to the integrin beta 2 subunit had only a slight effect, whereas treatment with mAb to the integrin alpha 4 and beta 2 subunits together abolished rolling interactions as well as arrest, and thus almost totally inhibited eosinophil accumulation. Our data indicate that P-selectin, but not E-selectin, is directly involved in eosinophil tethering on inflammatory endothelium while L-selectin mainly mediates intereosinophil interaction. VLA-4 has a crucial role in eosinophil arrest, and arrest is enhanced by exposure to chemoattractants.

Tonsillar B cells do not express PSGL-1, but a significant fraction displays the cutaneous lymphocyte antigen and exhibits effective E- and P-selectin ligand activity.

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM(+)/IgG(-) mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin alpha4beta7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as alpha4beta7.

Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins.

Antibodies immobilized on the wall of a flow chamber can support leukocyte rolling in shear flow. IgM mAb to Lewis(x) (CD15) and sialyl Lewis(x) (CD15s), which are carbohydrate antigens related to selectin ligands, plus mAb to CD48 and CD59, could mediate rolling. IgM and IgG mAb to L-selectin (CD62L), lymphocyte function-associated antigen 1 (CD11a), CD43, intercellular adhesion molecule 3 (CD50), and CD45 mediated only firm adhesion. In contrast to selectins, antibodies supported rolling only within a restricted range of site densities and wall shear stresses, outside of which firm adhesion or detachment occurred. When wall shear stress was increased, rolling velocity increased rapidly for antibodies but not for selectins. The kinetics of dissociation from the substrate of transiently tethered cells also increased more rapidly as a function of shear stress for antibodies than for selectins. These comparisons emphasize a number of remarkable features of selectins, including the lack of development of firm adhesion, and suggest that specialized molecular or cellular mechanisms must be required to explain their ability to support rolling over a wide range of environmental variables.