Quantitative Analysis of Different Cell Entry Routes of Actively Targeted Nanomedicines Using Imaging Flow Cytometry.

A rapid, high-throughput, and quantitative method for cell entry route characterization is still lacking in nanomedicine research. Here, we report the application of imaging flow cytometry for quantitatively analyzing cell entry routes of actively targeted nanomedicines. We first engineered ICAM1 antibody-directed fusogenic nanoliposomes (ICAM1-FusoNLPs) and ICAM1 antibody-directed endocytic nanolipogels (ICAM1-EndoNLGs) featuring highly similar surface properties but different cell entry routes: receptor-mediated membrane fusion and receptor-mediated endocytosis, respectively. By using imaging flow cytometry, we characterized their intracellular delivery into human breast cancer MDA-MB-231 cells. We found that ICAM1-FusoNLPs mediated a 2.8-fold increased cell uptake of fluorescent payload, FITC-dextran, with a 2.4-fold increased intracellular distribution area in comparison with ICAM1-EndoNLGs. We also investigated the effects of incubation time and endocytic inhibitors on the cell entry routes of ICAM1-FusoNLP and ICAM1-EndoNLG. Our results indicate that receptor-mediated membrane fusion is a faster and more efficient cell entry route than receptor-mediated endocytosis, bringing with it a significant therapeutic benefit in a proof-of-principle nanomedicine-mediated siRNA transfection experiment. Our studies suggest that cell entry route may be an important design parameter to be considered in the development of next-generation nanomedicines. © 2019 International Society for Advancement of Cytometry.

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: an atomic force microscopy study.

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha7 helix.

Structural analysis of human rhinovirus complexed with ICAM-1 reveals the dynamics of receptor-mediated virus uncoating.

Intercellular adhesion molecule 1 (ICAM-1) functions as the cellular receptor for the major group of human rhinoviruses, being not only the target of viral attachment but also the mediator of viral uncoating. The configurations of HRV3-ICAM-1 complexes prepared both at 4 degrees C and physiological temperature (37 degrees C) were analyzed by cryoelectron microscopy and image reconstruction. The particle diameters of two complexes (with and without RNA) representing uncoating intermediates generated at 37 degrees C were each 4% larger than that of those prepared at 4 degrees C. The larger virus particle arose by an expansive movement of the capsid pentamers along the fivefold axis, which loosens interprotomer contacts, particularly at the canyon region where the ICAM-1 receptor bound. Particle expansion required receptor binding and preceded the egress of the viral RNA. These observations suggest that receptor-mediated uncoating could be a consequence of restrained capsid motion, where the bound receptors maintain the viral capsid in an expanded open state for subsequent genome release.

ZO-1 redistribution and F-actin stress fiber formation in pulmonary endothelial cells after thermal injury.

BACKGROUND: In response to isolated inflammatory stimuli, changes in endothelial cell morphology that enhance paracellular flow of solutes result from F-actin stress fiber formation, myosin phosphorylation, and actin anchoring protein (ZO-1) modifications. We hypothesized that myosin light chain kinase inhibition would diminish burn-enhanced endothelial monolayer permeability by secondarily preventing F-actin and actin anchoring protein rearrangements. METHODS: Human pulmonary microvascular endothelial cells were treated for 4 hours with 20% human burn serum (isolated from patients with > 45% total body surface area thermal injury or healthy volunteers). Select cultures were pretreated with myosin light chain kinase inhibitors (ML-9). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine-phalloidin and anti-ZO-1 antisera and examined by means of confocal microscopy. RESULTS: Burn serum significantly enhanced monolayer permeability to albumin, whereas pretreatment with ML-9 limited this effect. Control cells maintained cortical F-actin and peripheral ZO-1 distributions (1a, b), whereas burn serum induced transcellular F-actin stress fiber formation and a diffuse ZO-1 staining (2a, b). ML-9 prevented burn-induced actin rearrangements, but not the diffuse redistribution of ZO-1. CONCLUSION: These data demonstrate that endothelial F-actin stress fiber formation and ZO-1 redistribution contribute to postburn loss of pulmonary endothelial monolayer integrity. Although myosin phosphorylation appears to be required for endothelial F-actin stress fiber formation, redistribution of actin-membrane anchoring proteins appears to be regulated independently after thermal injury.

Subcellular localization of intercellular adhesion molecule-1 in colonic mucosa in ulcerative colitis.

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells. In ulcerative colitis (UC), ICAM-1 is suggested also to be involved in the further migration of leukocytes toward the epithelial lining, and in colonic tissue it has been reported to be expressed by cell types other than endothelial cells. This study aimed at determining the ultrastructural localization of ICAM-1 on cells belonging to the colonic mucosa from patients with UC. Colonic biopsies from 3 UC patients and 3 control subjects were examined ultrastructurally by immunogold labeling of ICAM-1. ICAM-1 was expressed on the luminal cell membranes of endothelial cells in both controls and inflamed and noninflamed UC colon, although the density was significantly increased in UC (p < .0001). Labeling was observed on the basal endothelial cell membranes and on macrophages and plasma cells in inflamed UC colon only. Epithelial cells did not express ICAM-1. ICAM-1 appears to be constitutively upregulated on the luminal endothelial membrane in UC, and the expression on basal endothelial membranes in active UC only suggests that ICAM-1 is more extensively involved in the leukocyte migration than previously acknowledged.

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1).

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).

Fitting atomic models into electron-microscopy maps.

Combining X-ray crystallographically determined atomic structures of component domains or subunits with cryo-electron microscopic three-dimensional images at around 22 A resolution can produce structural information that is accurate to about 2.2 A resolution. In an initial step, it is necessary to determine accurately the absolute scale and absolute hand of the cryo-electron microscopy map, the former of which can be off by up to 5%. It is also necessary to determine the relative height of density by using a suitable scaling function. Difference maps can identify, for instance, sites of glycosylation, the position of which helps to fit the component structures into the EM density maps. Examples are given from the analysis of alphaviruses, rhinovirus-receptor interactions and poliovirus-receptor interactions.

Distinct cellular receptor interactions in poliovirus and rhinoviruses.

Receptor binding to human poliovirus type 1 (PV1/M) and the major group of human rhinoviruses (HRV) was studied comparatively to uncover the evolution of receptor recognition in picornaviruses. Surface plas- mon resonance showed receptor binding to PV1/M with faster association and dissociation rates than to HRV3 and HRV16, two serotypes that have similar binding kinetics. The faster rate for receptor association to PV1/M suggested a relatively more accessible binding site. Thermodynamics for receptor binding to the viruses and assays for receptor-mediated virus uncoating showed a more disruptive receptor interaction with PV1/M than with HRV3 or HRV16. Cryo-electron microscopy and image reconstruction of receptor-PV1/M complexes revealed receptor binding to the 'wall' of surface protrusions surrounding the 'canyon', a depressive surface in the capsid where the rhinovirus receptor binds. These data reveal more exposed receptor-binding sites in poliovirus than rhinoviruses, which are less protected from immune surveillance but more suited for receptor-mediated virus uncoating and entry at the cell surface.

Expression of adhesion molecules relevant to leukocyte migration on the microvilli of liver peritoneal mesothelial cells.

To help assess the immunological functions of the liver peritoneum, expression and 3D-microlocalization of adhesion molecules were studied by immuno-SEM and -TEM. The peritoneal tissues of the liver obtained from lipopolysaccharide (LPS, 1.5 microg/g BW for 24 hr)-stimulated (n = 18 including nine controls) and non-stimulated mice (n = 6 including three controls) were analyzed by immunolabeling with 15 nm gold particle single-labeling analysis of ICAM-1, ICAM-2, VCAM-1, MAdCAM-1, PECAM-1, ELAM-1, and CD105 expression. In addition, 10 and 20 nm gold particle double-labeling analysis of ICAM-1 and VCAM-1 was carried out with conventional TEM and BSE (backscatter electron) imaging. Gold particles detected in the peritoneal mesothelial cells were quantified using a computer analyzer, LUZEX III. Only ICAM-1 in non-stimulated mice and both ICAM-1 and VCAM-1 in LPS-stimulated mice were expressed on the mesothelium, but no other adhesion molecules were detected in either condition. Expression of ICAM-1 was consistently about four times greater than that of VCAM-1. Each adhesion molecule was restricted to the microvilli. ICAM-1 was expressed on all microvilli and tended to form clusters of three or four molecules. On the other hand, about 24% of the microvilli expressed VCAM-1 and less clustering was seen. Double-labeling techniques disclosed that VCAM-1 and ICAM-1 were rarely closely associated, usually spaced by about 40 nm. These results suggest that microvilli of the mesothelial cell play a significant role in leukocyte migration in the peritoneal cavity, by providing the important substrates for adhesion, ICAM-1 and VCAM-1.

Review: rhinoviruses and their ICAM receptors.

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen or macrophage-1 antigen. However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses.

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.

Abdominal peritoneum as a defense organ: analysis of ICAM-1 expression in the LPS-stimulated rat.

To investigate the immune environment of the peritoneal cavity, ICAM-1 (intercellular adhesion molecule) expression on the apical surface of the hepatic peritoneum of LPS (lipopolysaccharide) stimulated rats was analyzed ultrastructurally and chronologically with immunoTEM&SEM. ICAM-1 expression was restricted to the side of microvilli of the mesothelial cells. Microvilli demonstrated bulbous tips and included fuzzy coats and strands. Bulbous tips sometimes expressed the antigen, but fuzzy coats and strands did not. Intervillar cell surfaces lacked its expression. Although ICAM-1 expression increased eightfold 24 hr after stimulation, the selective expression remained unchanged. These results suggest that microvilli are closely associated with cell migration in the peritoneal cavity through adhesion molecules that establish a road for migration.

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand.

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Murine thymic nurse cells express ICAM-1 on caveolar and vacuolar membranes.

The thymic nurse cell is a unique type of epithelial cell in the thymic cortex. It is in intimate contact with the developing thymocytes by harbouring up to 200 thymocytes in distinct vacuoles, called caveoles. This investigation is concerned with the nurse cell expression of the intercellular adhesion molecule ICAM-1, the ligand for thymocyte LFA-1. Nurse cells from young Balb/c mice were isolated in a density gradient. ICAM-1 expression was studied by using two different immunotechniques: alkaline phosphatase labelled cryosections, and immunogold electron microscopy. The specific antibody was a monoclonal rat anti-mouse ICAM-1. Immunostaining of cryosections demonstrated that ICAM-1 is expressed on the surface membrane and in the internal caveolar membranes of thymic nurse cells. Electron microscopy of immunogold labelled sections revealed ICAM-1 on the surface membrane of thymic nurse cells and on the membranes of the caveoles, the small cytoplasmic vesicles, as well as on the Golgi apparatus.

Precise ultrastructural localization of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in patients with IgA nephropathy.

Using light and electron microscopy, we performed an immunohistochemical study of endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in 15 patients with IgA nephropathy to clarify the localization of these adhesion molecules. The normal portions of 2 kidneys removed due to localized carcinoma and 3 biopsies from patients without glomerular disease were used as a control. By light microscopy, ELAM-1, VCAM-1, and ICAM-1 all showed positive staining in IgA nephropathy, with the intensity of staining following the sequence ICAM-1 > VCAM-1 > ELAM-1. ELAM-1 and VCAM-1 showed a patchy distribution of moderate staining in the tissues, including the mesangium, crescents, adhesions, and tubules. In contrast, there was marked linear ICAM-1 staining throughout the vascular walls. ELAM-1 and VCAM-1 were positive on the basolateral surfaces of a few proximal tubular epithelial cells in association with inflammatory cell infiltration, while ICAM-1 was found on the brush border. ICAM-1 was positive in the glomerular capillary walls and interstitial vessels of the control kidney tissue, while ELAM-1 and VCAM-1 were virtually absent. By electron microscopy, ELAM-1 positivity on the urinary surface of the parietal/visceral epithelial cells was often associated with adherent mononuclear cells in the urinary space. VCAM-1 positivity was increased in the perinuclear space and/or cytoplasm of mesangial cells as well as at the mesangial cell-endothelial cell interface. These findings suggest that ELAM-1 and VCAM-1 may be more closely related than ICAM-1 to the major histopathological changes occurring in IgA nephropathy, including mesangial expansion, formation of crescents and adhesions, and tubulointerstitial injury.

Alopecia areata: immunohistochemistry and ultrastructure of infiltrate and identification of adhesion molecule receptors.

BACKGROUND: Alopecia areata (AA) is a noncicatricial alopecia with still unknown pathogenesis, but increasing evidence suggests that an immunologic process might be responsible for the disease. MATERIALS AND METHODS: Nineteen patients with AA were studied with ten of them in the progressive phase of the disease and nine in the stabilized phase. Biopsies of both affected and unaffected skin were taken. For immunohistochemistry, monoclonal antibodies directed against CD3, CD4, CD8, CD10a, CD36, and HLA-DR antigens, were used, as well as antibodies directed against adhesion molecules ICAM-1, ELAM-1 and LFA-1. For electron microscopy (EM), specimens were fixed in glutaraldehyde-sodium cacodylate buffer, post-fixed in osmium tetroxide, and stained with uranyl acetate. For statistical analysis, sections from involved and uninvolved skin of each patient for each antibody, the sign test, Fisher's F-test, and the Tukey-Kramer test were used. RESULTS: There was a rich infiltrate of CD4+ cells and CD1a+ cells, particularly in the perivascular zone of both unaffected and affected skin (here in the perivascular and in the peribulbar zone) in the progressive phase of AA. In the stabilized phase the infiltrate was scant, both in unaffected and affected skin and limited to the peribulbar area. Receptors of adhesion molecules (ICAM-2, ELAM-1, LFA-1) were strongly expressed, mainly at the microvascular level in both unaffected and affected skin in the progressive phase, but were only weakly or not at all expressed in the stabilized phase, again in unaffected and affected skin. Ultrastructural data confirmed the immunohistochemical findings and showed close contacts between infiltrating lymphocytes and Langerhans'-lineage cells mainly in the progressive phase. CONCLUSIONS: Our results suggest that: 1) an immunologic process, apparently carried out by CD4+ lymphocytes and by dendritic CD1a+ and CD36+ cells, may play a key role at least in the early phase of the disease involving primarily microvessels and later on the bulbar area; 2) the expression of adhesion molecule receptors is involved at the beginning of the disease by mediating the adherence of leukocytes to endothelial cells and subsequent trafficking into the dermis.

Ultrastructural localization of the intercellular adhesion molecule (ICAM-1) on the cell surface of high endothelial venules in lymph nodes.

BACKGROUND: The high endothelial venules (HEV) in the lymph nodes are essential for lymphocyte recirculation. As a first step, the HEV surface interacts with lymphocytes through adhesion molecules. It is important to know where adhesion molecules are expressed on the surface ultrastructure and how these structures interact with lymphocytes. METHODS: To demonstrate the ultrastructural mechanism of interaction between the HEV surface and lymphocytes through the intercellular adhesion molecule (ICAM-1), rat mesenteric lymph nodes were perfused through the superior mesenteric artery with the primary antibody (antirat ICAM-1 antibody) and secondary antibody (antimouse IgG coupled to 15 nm gold particles), which were diluted with hypothermic University of Wisconsin (UW) solution. After the immunoreaction, we analyzed the HEV three-dimensionally and quantitatively using immunoscanning electron microscopy (ISEM) combined with transmission electron microscopy (TEM). RESULTS: HEV expressed ICAM-1 in a 5-30-fold higher concentration than other vessels. Its distribution was extensive over the luminal surface of the cell down to the junctional area. The endothelial surface of HEV undulated to form branched microfolds, along which ICAM-1 was expressed. Cytoplasmic processes of lymphocytes were seen in microfurrows between microfolds and adhered to the sides of the folds. CONCLUSIONS: These observations imply that the microfolds expressing ICAM-1 and microfurrows are specific ultrastructural features for trapping lymphocytes, thus initiating lymphocyte emigration into the lymph node parenchyma.

Early expression of intercellular adhesion molecule-1 in the corneal endothelium stimulated by endotoxin: an immuno-scanning electron microscopical study.

Three-dimensional localization of the intercellular adhesion molecule-1 (ICAM-1) in the corneal endothelium stimulated by Salmonella typhimurium endotoxin was investigated using immuno-scanning electron microscopy (SEM). Samonella typhimurium endotoxin, 100 micrograms, was injected in Lewis rats weighing 200 grams. The animals, including the controls, were sacrificed and both eyes enucleated at 0, 3, 12 and 24 hours after injection (n = 3 each time). After resection, the corneas were immersed in hypothermic University of Wisconsin solution with monoclonal mouse-anti-rat ICAM-1 IgG and then goat-anti-mouse IgG coupled to 15 nm gold particles. Then the corneas were prepared conventionally for scanning electron microscopy. Histotopographical examination with immuno-SEM revealed that ICAM-1 antigen increased on the corneal endothelium by 3 hours postinjection. The particles were arranged along the cytoplasmic processes, especially at the summits. The number of particles was 3.3 +/- 0.8/microns2 in the control, 3.6 +/- 0.8/microns2 at 0 hour, 14.4 +/- 0.9/microns2 at 3 hours, 25.4 +/- 1.4/microns2 at 12 hours, and 22.7 +/- 2.6/microns2 at 24 hours postinjection. ANOVA indicated that the time-course was an important factor (P < 0.01). Our results showed that ICAM-1 could be augmented in the corneal endothelium by endotoxin. The interrelationship between ICAM-1 expression and cytoplasmic processes seems to be important for the neutrophil-binding mechanism.