Intercellular adhesion molecule-1 (ICAM-1) in the mouse facial motor nucleus after axonal injury and during regeneration

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1–2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4–7 days. The second induction of ICAM-1 occured at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions

Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling.     

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4⁺ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4⁺ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3^−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2^−/− (recombination activating gene-2-deficient) mice adoptively transferred CD4⁺ T-cells isolated from CCR3^−/− mice, but not in CCR3^−/− mice adoptively transferred CD4⁺ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4⁺ T-cell- and CCR3-mediated neuroprotection after FMN injury.     

ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into the alveolar space

Neutrophil recruitment into lung constitutes a major response to airborne endotoxins. In many tissues endothelial intercellular adhesion molecule-1 (ICAM-1) interacts with lymphocyte function associated antigen-1 (LFA-1) on neutrophils, and this interaction plays a critical role in neutrophil recruitment. There are conflicting reports about the role of ICAM-1 in neutrophil recruitment into lungs. We studied neutrophil recruitment into alveolar space in a murine model of aerosolized LPS-induced lung inflammation. LPS induces at least a 100-fold increase in neutrophil numbers in alveolar space, as determined by flow cytometry of bronchoalveolar lavage fluid. Neutrophil recruitment was reduced by 54% in ICAM-1 null mice and by 45% in LFA-1 null mice. In wild-type mice treated with anti-ICAM-1 and anti-LFA-1 antibodies, there was 51 and 58% reduction in the neutrophil recruitment, respectively. In chimeric mice, generated by the transplantation of mixtures of bone marrows from LFA-1 null and wild-type mice, the normalized recruitment of LFA-1 null neutrophils was reduced by 60% compared with wild-type neutrophils. Neither the treatment of ICAM-1 null mice with a function-blocking antibody to LFA-1 nor the treatment of LFA-1 null mice with anti-ICAM-1 antibody resulted in further reduction in the recruitment compared with untreated ICAM-1 null and LFA-1 null mice. We conclude that ICAM-1 and LFA-1 play critical roles in the recruitment of neutrophils into the alveolar space in aerosolized LPS-induced lung inflammation, and LFA-1 serves as a ligand of ICAM-1 in the lung.     

Detection of ICAM-1 in experimentally induced colitis of ICAM-1-deficient and wild-type mice: an immunohistochemical study

Adhesion molecules (e.g. ICAM-1, CD 54) are known to be upregulated on activated vascular endothelial cells during inflammatory reactions. To study the role of ICAM-1 in intestinal inflammation in vivo, we induced acute experimental colitis in wild-type (C57BL/6) mice and ICAM-1-deficient mice, by feeding the animals with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. In the control strain the immunohistochemical staining showed a very pronounced endothelial upregulation of ICAM-1 after the DSS treatment observed in areas of inflammatory infiltrate, especially in venules or arterioles of the propria and submucosa, and partly in the mesocolon. DSS-fed ICAM-1-deficient mice showed no endothelial enhancement and only faint staining of venules or capillaries approaching that encountered in the control ICAM-1-deficient animals. Our data indicate that ICAM-1 may play a crucial role in the development of acute intestinal inflammation, consistent with our finding that ICAM-1 deficiency can obviate severe forms of experimentally induced colitis in mice.     

Absence of allograft ICAM-1 attenuates alloantigen-specific T cell priming,but not primed T cell trafficking into the graft,to mediate acute rejection

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1(-/-) donors were tested. Wild-type C57BL/6 (H-2(b)) heart allografts were rejected by A/J (H-2(a)) recipients on days 7-9, whereas B6.ICAM-1(-/-) allografts survived until days 18-23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4(+) and CD8(+) T cells in the ICAM-1(-/-) allografts were 20-30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-gamma from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1(-/-) mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1(-/-) allografts on days 5-6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.     

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.     

Intercellular adhesion molecule 1 deficiency leads to impaired recruitment of T lymphocytes and enhanced host susceptibility to infection with Trypanosoma cruzi

In this study, we investigated the involvement of Th1 cytokines in the expression of cell adhesion molecules (CAM) and recruitment of inflammatory cells to the heart of mice infected with Trypanosoma cruzi. Our results show that endogenously produced IFN-gamma is essential to induce optimal expression of VCAM-1 and ICAM-1 on the cardiac vascular endothelium of infected mice. Furthermore, the influx of inflammatory cells into the cardiac tissue was impaired in Th1 cytokine-deficient infected mice, paralleling the intensity of VCAM-1 and ICAM-1 expression on the vascular endothelium. Consistent with the importance of ICAM-1 in host resistance, ICAM-1 knockout (KO) mice were highly susceptible to T. cruzi infection, as assessed by mortality rate, parasitemia, and heart tissue parasitism. The enhanced parasitism was associated with a decrease in the numbers of CD4(+) and CD8(+) T lymphocytes in the heart tissue of ICAM-1 KO mice. Additionally, ICAM-1 KO mice mounted an unimpaired IFN-gamma response and IFN-gamma-dependent production of reactive nitrogen intermediates and parasite- specific IgG2a. Supporting the participation of ICAM-1 in cell migration during T. cruzi infection, the entrance of adoptively transferred PBL from T. cruzi-infected wild-type C57BL/6 mice into the cardiac tissue of ICAM-1 KO mice was significantly abrogated. Therefore, we favor the hypothesis that ICAM-1 plays a crucial role in T lymphocyte recruitment to the cardiac tissue and host susceptibility during T. cruzi infection.     

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up-regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over-expression of P311 induces p21(WAF1/Cip1) expression, leading PC12 cells to differentiate and to have neuron-like morphologies. Adenovirus-mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.     

ICAM-1 and beta2 integrin deficiency impairs fat oxidation and insulin metabolism during fasting

Intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins play critical roles in immune responses. ICAM-1 may also participate in regulation of energy balance because ICAM-1-deficient mice become obese on a high-fat diet. We show that mice deficient in these adhesion receptors are unable to respond to fasting by up-regulation of fatty acid oxidation. Normal mice, when fasted, exhibit reduced circulating neutrophil counts and increased ICAM-1 expression and neutrophil recruitment in liver. Mice lacking ICAM-1 or beta2 integrins fail to show these responses--instead they become hypoglycemic with steatotic livers. Fasting ICAM-1-deficient mice reduce insulin more slowly than wild-type mice. This produces fasting hyperinsulinemia that prevents activation of adenosine mono-phosphate (AMP)-activated protein kinase in muscles and liver, which results in decreased import of long chain fatty acids into mitochondria. Thus, we show a new role for immune cells and their adhesion receptors in regulating metabolic response to fasting.     

Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration

To examine the role of the ICAM-1 C-terminal domain in transendothelial T lymphocyte migration and ICAM-1-mediated signal transduction, mutant human (h)ICAM-1 molecules were expressed in rat brain microvascular endothelial cells. The expression of wild-type hICAM-1 resulted in a significant increase over basal levels in both adhesion and transendothelial migration of T lymphocytes. Endothelial cells (EC) expressing ICAM-1 in which the tyrosine residue at codon 512 was substituted with phenylalanine (hICAM-1(Y512F)) also exhibited increased lymphocyte migration, albeit less than that with wild-type hICAM-1. Conversely, the expression of truncated hICAM-1 proteins, in which either the intracellular domain was deleted (hICAM-1DeltaC) or both the intracellular and transmembrane domains were deleted through construction of a GPI anchor (GPI-hICAM-1), did not result in an increase in lymphocyte adhesion, and their ability to increase transendothelial migration was attenuated. Truncated hICAM-1 proteins were also unable to induce ICAM-1-mediated Rho GTPase activation. EC treated with cell-permeant penetratin-ICAM-1 peptides comprising human or rat ICAM-1 intracellular domain sequences inhibited transendothelial lymphocyte migration, but not adhesion. Peptides containing a phosphotyrosine residue were equipotent in inhibiting lymphocyte migration. These data demonstrate that the intracellular domain of ICAM-1 is essential for transendothelial migration of lymphocytes, and that peptidomimetics of the ICAM-1 intracellular domain can also inhibit this process. Such competitive inhibition of transendothelial lymphocyte migration in the absence of an affect on adhesion further implicates ICAM-1-mediated signaling events in the facilitation of T lymphocyte migration across brain EC. Thus, agents that mimic the ICAM-1 intracellular domain may be attractive targets for novel anti-inflammatory therapeutics.     

Inhibition of Rho GTPases with protein prenyltransferase inhibitors prevents leukocyte recruitment to the central nervous system and attenuates clinical signs of disease in an animal model of multiple sclerosis

The ICAM-1-mediated brain endothelial cell (EC)-signaling pathway induced by adherent lymphocytes is a central element in facilitating lymphocyte migration through the tight endothelial barrier of the brain. Rho proteins, which must undergo posttranslational prenylation to be functionally active, have been shown to be an essential component of this signaling cascade. In this study, we have evaluated the effect of inhibiting protein prenylation in brain ECs on their ability to support T lymphocyte migration. ECs treated in vitro with protein prenylation inhibitors resulted in a significant reduction in transendothelial T lymphocyte migration. To determine the therapeutic potential of this approach, an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, was induced in Biozzi ABH mice. Animals treated before disease onset with protein prenylation inhibitors exhibited a dramatic and significant reduction in both leukocyte infiltration into the CNS and clinical presentation of disease compared with untreated animals. These studies demonstrate, for the first time, the potential for pharmacologically targeting CNS EC signaling responses, and particularly endothelial Rho proteins, as a means of attenuating leukocyte recruitment to the CNS.     

SOCS1 regulates CCR7 expression and migration of CD4+ T cells into peripheral tissues

Suppressors of cytokine signaling (SOCS) proteins control many aspects of lymphocyte function through regulation of STAT pathways. SOCS1-deficient mice develop severe skin and eye diseases that result from massive infiltration of inflammatory cells into these tissues. In this study, we have used SOCS1-, STAT1-, or STAT6-deficient mice, as well as, T cells with stable overexpression or deletion of SOCS1, to examine whether SOCS1 is involved in regulating lymphocyte trafficking to peripheral tissues. We show that SOCS1-deficient mice have increased numbers of T cells with characteristics of effector memory cells and expression of CCR7, a protein that promotes retention of T cells in lymphoid tissues, is markedly reduced in these cells. The decrease in CCR7 expression correlates with hyperactivation of STAT6, suggesting that aberrant recruitment of T cells into SOCS1-deficient mouse skin or eye results from abrogation of negative feedback regulation of STAT6 activation and CCR7 expression. Consistent with in vivo regulation of CCR7 expression and lymphocyte migration by SOCS1, forced overexpression of SOCS1 in T cells up-regulates CCR7 expression and enhances chemotaxis toward CCL19 or CCL21. CCR6 and CXCR3 are also up-regulated on SOCS1-deficient T cells and in situ analysis of the cornea or retina further reveal that these cells may mediate the chronic skin and eye inflammation through recruitment of Th1 and Th17 cells into these tissues. Collectively, these results suggest that SOCS1 regulates steady-state levels of chemokine receptors through its inhibitory effects on STAT pathways and this may underscore its role in regulating recruitment and retention of effector cells into nonlymphoid tissues.     

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

ICAMs Redistributed by Chemokines to Cellular Uropods as a Mechanism for Recruitment of T Lymphocytes

The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte–endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5–10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0⁺ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti– ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.     

Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking

The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1. The entry of lymphocytes into peripheral and mesenteric lymph nodes was found to be unaffected by the functional deletion of either ICAM-1 or ICAM-2. However, when both pathways were blocked, recirculation through lymph nodes was strongly reduced. Trapping of lymphocytes in the lung after i.v. injection is partly mediated by LFA-1/ICAM interactions; the data presented in this study show an exclusive role of ICAM-1 in LFA-1-dependent lung trapping. Similarly, ICAM-1, but not ICAM-2, was required for the migration of T effector cells into the inflamed skin. These results indicate that ICAM-1 and ICAM-2 have redundant functions in lymphocyte recirculation through lymph nodes, but ICAM-1 is unique in supporting migration into inflamed sites and trapping within the lung.