If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
To whom correspondence should be addressed: Division of Infectious Diseases, University of British Columbia, Rm. 452D, 2733 Heather St., Vancouver, British Columbia V5Z 3J5, Canada. Tel.: 604-875-4011; Fax: 604-875-4013;
Department of Medicine (Division of Infectious Diseases) and theDepartment of Microbiology and Immunology, The University of British Columbia, Faculties of Medicine and Science, The Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia V5Z 3J5, Canada and
* This work was supported by Medical Research Council of Canada Grant MT-8633.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Mechanisms regulating lipopolysaccharide (LPS)-induced adherence to intercellular adhesion molecule (ICAM)-1 were examined using THP-1 cells transfected with CD14-cDNA (THP-1wt). THP-1wt adherence to ICAM-1 was LPS dose-related, time-dependent, and inhibited by antibodies to either CD14 or leukocyte function associated antigen (LFA)-1, but was independent of any change in the number of surface expressed LFA-1 molecules. A potential role for phosphatidylinositol (PI) 3-kinase (PI 3-kinase) in LPS-induced adherence was examined using the PI 3-kinase inhibitors LY294002 and Wortmannin. Both inhibitors selectively attenuated LPS-induced, but not phorbol 12-myristate 13-acetate-induced adherence. Inhibition by these agents was unrelated to any changes in either LPS binding to or LFA-1 expression by THP-1wt cells. LPS-induced adherence was also abrogated in U937 cells transfected with a dominant negative mutant of of PI 3-kinase. Toxin B from Clostridium difficile, an inhibitor of the Rho family of GTP-binding proteins, abrogated both PI-3 kinase activation and adherence induced by LPS. Cytohesin-1, a phosphatidylinositol 3,4,5-triphosphate-regulated adaptor molecule for LFA-1 activation, was found to be expressed in THP-1wt cells. In addition, treatment of THP-1wt with cytohesin-1 antisense attenuated LPS-induced adherence. These findings suggest a model in which LPS induces adherence through a process of “inside-out” signaling involving CD14, Rho, and PI 3-kinase. This converts low avidity LFA-1 into an active form capable of increased binding to ICAM-1. This change in LFA-1 appears to be cytohesin-1-dependent.
Adherence of monocytes to endothelial cells is an essential requirement for the localization of these cells to sites of tissue inflammation (
). This is consistent with a process of “inside-out” signaling that converts LFA-1 into an activated form capable of mediating increased adhesion. It is important to note that conditions which give rise to increased adherence do not necessarily lead to increased cell surface expression of LFA-1 (
The signaling events that link cell stimulation to the activation of LFA-1 are incompletely understood. Recently, a regulatory protein that interacts with the cytoplasmic tail of CD18 has been cloned (
). This protein, cytohesin-1, contains a pleckstrin homology domain that binds the phosphatidylinositol 3-kinase (PI 3-kinase) metabolite, phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3), leading to changes in properties of the protein (
). Taken together, these findings suggest the possibility that LPS-induced adhesion may be mediated through a pathway involving PI 3-kinase leading to changes in LFA-1 activity. The results of the present study show that LPS binding to CD14 induces monocyte adherence dependent upon LFA-1, ICAM-1, and cytohesin-1, via a PI 3-kinase-dependent pathway regulated by the small GTP-binding protein Rho.
This study examined signaling events required for LPS-induced adherence. The system used involved a quantitative, microtiter adhesion assay, CD14 transfected THP1 cells, and immobilized sICAM-1. Adherence in this system was found to be dependent upon CD14 (Fig. 2). Experiments that examined competitive inhibition of LPS-induced adherence using mAbs to CD14, CD18, and CD11a (Fig. 3A) provided direct evidence that LPS-induced adherence to sICAM-1 involves a CD14 mediated signal leading to activation of cell surface expressed LFA-1. These findings are consistent with previous data showing that antibody cross-linking of cell surface CD14 induces LFA-1 activation (
). LPS effects on LFA-1 did not involve changes in the expression of CD18 or CD11a. This indicates that LPS-induced adhesion was related to increased affinity of LFA-1 for ICAM-1 rather than to increased expression of cell surface LFA-1. Such changes in the properties of LFA-1 are presumably mediated by a specific pathway of inside-out signaling initiated through CD14.
The requirement for PI 3-kinase activity in a variety of leukocyte functions, together with its apparent role in the adhesion of platelets (
), made this enzyme an attractive candidate for mediating signaling through CD14 for monocyte adhesion. This hypothesis was supported further by the finding that LPS induces the CD14-dependent association of an activated form PI 3-kinase with p53/p56lyn (
). The role of PI 3-kinase in LPS-induced adherence was examined using two different approaches. The first involved the use of two structurally unrelated PI 3-kinase inhibitors wortmannin and LY294002. LPS-induced adherence was attenuated by both of these agents (Fig. 4). The effects of wortmannin are considered to be relatively specific for PI 3-kinase at concentrations similar to those used in this study (50 nm, Fig. 4). However, the compound has been shown to inhibit phospholipase A2 with an IC50 similar to that reported previously for PI 3-kinase (
). The findings that both compounds inhibited LPS-induced adherence, therefore, support the argument that PI 3-kinase is involved in the regulation of adherence in response to LPS. This conclusion is supported further by experiments in which a dominant negative mutant of PI 3-kinase (Δp85) expressed in U937 cells completely abrogated LPS-induced adherence to sICAM-1 (Fig. 5B). It has been shown previously that incubation of monocytes with LPS activates PI 3-kinase, leading to increased cellular levels of PtdIns-3,4,5-P3 (
). This finding suggested the possibility that LPS-induced adherence may be Rho-regulated and mediated by PI 3-kinase. In this report, a requirement for Rho in LPS-induced adherence was suggested by studies that used C. difficile toxin B, which specifically inhibits Rho family proteins (
). Pretreatment of THP-1wt cells with toxin B for 30 min. attenuated LPS-induced adherence to sICAM-1 (Fig. 6,A and B). In contrast, PMA-induced adherence appeared to be mediated by a toxin B-insensitive pathway (Fig. 6,A and B). This dichotomy is consistent with reports showing that PMA-induced responses in a variety of cell types may be either resistant to Rho toxins or that inhibition of these responses requires prolonged periods of incubation with toxins (24 h and more) (
). For example 8–24-h of pretreatment with botulinum C3 exoenzyme, another inhibitor of Rho family proteins, was required to attenuate PMA-induced, LFA-1/ICAM-1-dependent aggregation of the lymphoblastoid cell line JY (
). In the present study, THP-1 cells were incubated with toxin B for up to a maximum of 3 h. Under these conditions it is clear that adherence induced by LPS was toxin-sensitive, whereas the response to PMA was markedly resistant.
The findings that both Rho and PI 3-kinase appeared to be essential for LPS-induced adherence raised the question as to whether they act independently or whether they are positioned together in a single signaling pathway. Fig. 6C shows that toxin B prevented activation of PI 3-kinase in LPS-stimulated THP-1wt cells, suggesting that Rho regulates this LPS response in monocytes. This observation is consistent with previous reports showing involvement of Rho in PI 3-kinase activation in other systems (
). Although we cannot completely eliminate the possibility of a direct, PI 3-kinase-independent role for Rho in regulating monocyte adherence, the data suggest that LPS triggers Rho-mediated activation of PI 3-kinase, leading to downstream effects on LFA-1 and monocyte adherence.
An important question arising from these observations is how PI 3-kinase activation modulates the properties of LFA-1. Recently, cytohesin-1 has been shown to interact with the cytoplasmic tail of CD18 (
). Because a suitable antibody to cytohesin-1 was not available, RT-PCR of THP-1 mRNA was used to examine whether cytohesin-1 is expressed in THP-1 cells. The results shown in Fig. 7 confirmed that the cytohesin-1 gene is transcribed under basal conditions. To directly address the role of cytohesin-1 in LPS-induced adherence, cytohesin-1-specific antisense oligonucleotides were used. To ensure maximal specificity of the antisense oligonucleotide, the sequence selected was from a region lacking significant homology with other sequenced human genes. The oligonucleotides were also phosphorothioate-modified to limit degradation and purified by high performance liquid chromatography to remove incomplete synthesis products. In addition fluorescein-modified antisense and FACS analysis were used to monitor oligonucleotide incorporation into cells. The finding that antisense treatment of THP-1 cells, but not treatment with sense oligonucleotide, significantly attenuated LPS-induced adherence to ICAM-1 (Fig. 8A) provided compelling evidence to suggest that cytohesin-1 plays an essential role in adherence induced by LPS. Of note, the proportion of cells that incorporated antisense-oligonucleotide (Fig. 8B) correlated closely with the fraction of cells that failed to adhere in response to LPS (Fig. 8A).
Taken together, the results presented are consistent with a model (Fig.9) in which LPS binding to CD14 switches on the small G-protein Rho leading to activation of PI 3-kinase. This leads to increased monocyte adherence, dependent upon changes in the adhesive properties of LFA-1. The latter appears to involve the interaction of PtdIns-3,4,5-P3, cytohesin-1, and LFA-1.
We thank Dr. J. R. Woska, Jr. for sICAM-1, Dr. Glen Armstrong for toxin B, Dr. W. C. Van Voorhis for antibody to CD14, and Dr. R. Ulevitch for THP-1wt and THP-1rsv cells.