The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage

CD14 is a myeloid differentiation Ag expressed primarily on peripheral blood monocytes and macrophages. Although its function is unknown, the CD14 gene maps to a region encoding several myeloid growth factors and receptors. Analysis of the CD14 protein sequence deduced from the cDNA shows that although the CD14 protein contains a characteristic leader peptide, it lacks a characteristic transmembrane region, suggesting that CD14 may be anchored to the membrane via glycosylphosphatidylinositol (PI). Treatment of monocytes as well as a CD14-expressing neuroglioma cell line with PI-phospholipase C removed CD14 from the cell surface. Furthermore, monocytes from a patient with paroxysmal nocturnal hemoglobinuria, a disease characterized by lack of expression of other PI-linked proteins, failed to express CD14. Interestingly, the CD14-expressing neuroglioma cell line, which had been transfected with a single CD14 cDNA, released a soluble form of CD14 into the supernatant. Soluble forms of CD14 have previously been observed in serum of normal individuals and in culture supernatants of CD14+ cells. Biosynthetic experiments reveal that this soluble form of CD14 (48 kDa), which is smaller than the form released from the membrane by PI-phospholipase C (53 kDa), does not contain ethanolamine, the first constitutent of the PI-anchoring system. These studies demonstrate that CD14 is a member of the family of PI-anchored proteins and suggest that soluble forms of CD14 represent molecules that completely lack the PI-anchoring system.     

Interleukin-10 in combination with M-CSF and IL-4 contributes to development of the rare population of CD14+CD16++ cells derived from human monocytes

Peripheral blood CD14(+)CD16(++) monocytes (Mo) are a rare Mo subpopulation known to undergo expansion in various diseases. We show here that IL-10 in the presence of M-CSF and IL-4 triggers the generation of CD14(+)CD16(++) cells from highly purified human cord blood (CB) and adult blood Mo. CB Mo were more sensitive to this cytokine combination than adult Mo. IL-10-induced CD14(+)CD16(++) cells that expressed dendritic cell markers: CD80, CD86, HLA-DR, and CD83 and initiated significantly decreased allogeneic mixed lymphocyte reactions (MLRs). Blockage of CD86, but not CD80, further down-modulated MLRs induced by CD14(+)CD16(++)cells. CD14(+)CD16(++) cells had similar features to CD14(+)CD16(++) Mo in that they expressed increased level of CCR5, efficiently produced TNF-alpha, and displayed higher MLR than CD14(+)CD16(-) Mo. Together, these results demonstrate that M-CSF, IL-4, and IL-10 drive Mo into CD14(+)CD16(++) cells similar to those identified in vivo, and CB Mo, due to their increased responsiveness, may be a useful starting cell source to study differentiation of CD14(+)CD16(++) cells.     

Influence of CD14 on ligand interactions between lipopolysaccharide and its receptor complex

The interaction of LPS (endotoxin) with the CD14-TLR4 receptor complex modulates the host innate immune response. Several studies using partial structures of LPS have suggested that TLR4 determines the ligand specificity of this complex, and that CD14 indiscriminately serves to deliver the ligand to TLR4. This conclusion has been made despite observations that the response of TLR4(+/+),CD14(-/-) macrophages to LPS is very weak. To determine whether CD14 itself plays a role in specific ligand recognition, the influences of various partial structures of LPS on induction of the proinflammatory cytokine, TNF, by CD14(+/+) and CD14(-/-) macrophages were compared. These studies show that the ligand specificities of CD14(+/+) and CD14(-/-) macrophages are very different. When CD14 is present, the receptor complex shows exquisite specificity for smooth LPS, the major form expressed by Gram-negative bacteria; however, as increasing amounts of carbohydrate are removed from smooth LPS, the sensitivity of CD14(+/+) macrophages decreases as much as 500-fold. In contrast, CD14(-/-) macrophages are unable to distinguish between smooth LPS and its various partial structures. Furthermore, CD14(-/-) macrophages are 150,000-fold less sensitive than CD14(+/+) macrophages to smooth LPS. A similar ability to distinguish the differing LPS structures of various bacteria such as Bacteroides fragilis and Salmonella abortus are observed for CD14(+/+), but not CD14(-/-), macrophages. Thus, CD14(+/+), but not CD14(-/-), macrophages are highly sensitive to stimulation by natural forms of LPS and show the ability to distinguish between various LPS ligands, consistent with CD14 being a highly specific receptor.     

Diverging pathways for lipopolysaccharide and CD14 in human monocytes

BACKGROUND: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. METHODS: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surface-biotinylated cells with LPS at 37 degrees C or 4 degrees C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37 degrees C or at 4 degrees C) used, indicating that these CD14 molecules were not taken up by an active process. CONCLUSIONS: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface.     

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.     

Surfactant proteins A and D bind CD14 by different mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387-395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.     

GPI-linked endothelial CD14 contributes to the detection of LPS

The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR- and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only approximately 30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment.     

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.     

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes

Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied. To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M. tuberculosis expressing green fluorescent protein. Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14. However, most internalized bacteria were found in CD14+ but not in CD14- monocytes because M. tuberculosis infection upregulated CD14 expression. We conclude that: (1) CD14 does not mediate cellular entry by M. tuberculosis; (2) M. tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response.     

Divergent response to LPS and bacteria in CD14-deficient murine macrophages

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.     

Negative Regulation of Toll-like Receptor-4 Signaling through the Binding of Glycosylphosphatidylinositol-anchored Glycoprotein,CD14, with the Sialic Acid-binding Lectin,CD33

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.     

CD14<Superscript>++</Superscript>CD16<Superscript>−</Superscript> and CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> human monocyte adhesion to endothelial cells

Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14⁺CD16⁺ and CD14⁺⁺CD16⁻ monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14⁺CD16⁺ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14⁺⁺CD16⁻ monocytes. Adhesion of CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did not affect the adhesion of monocytes. These results show that CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes can be recruited to the inflamed endothelium, but CD14⁺CD16⁺ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14⁺⁺CD16⁻ monocytes.     

Signaling through CD14 attenuates the inflammatory response to Borrelia burgdorferi, the agent of Lyme disease

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14(-/-) mice exhibited more severe and persistent inflammation than did CD14(+/+) mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14(-/-) mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14(-/-) macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.     

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.     

Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

Mucosal and peripheral Lin- HLA-DR+ CD11c/123- CD13+ CD14- mononuclear cells are preferentially infected during acute simian immunodeficiency virus infection

Massive infection of memory CD4 T cells is a hallmark of early simian immunodeficiency virus (SIV) infection, with viral infection peaking at day 10 postinfection (p.i.), when a majority of memory CD4 T cells in mucosal and peripheral tissues are infected. It is not clear if mononuclear cells from the monocyte and macrophage lineages are similarly infected during this early phase of explosive HIV and SIV infections. Here we show that, at day 10 p.i., Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(-) macrophages in the jejunal mucosa were infected, albeit at lower levels than CD4 memory T cells. Interestingly, Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(-) macrophages in peripheral blood, like their mucosal counterparts, were preferentially infected compared to Lin(-) HLA-DR(+) CD11c/123(-) CD13(+) CD14(+) monocytes, suggesting that differentiated macrophages were selectively infected by SIV. CD13(+) CD14(-) macrophages expressed low levels of CD4 compared to CD4 T cells but expressed similar levels of CCR5 as lymphocytes. Interestingly, CD13(+) CD14(-) macrophages expressed Apobec3G at lower levels than CD13(+) CD14(+) monocytes, suggesting that intracellular restriction may contribute to the differential infection of mononuclear subsets. Taken together, our results suggest that CD13(+) CD14(-) macrophages in mucosal and peripheral tissues are preferentially infected very early during the course of SIV infection.     

Engagement of the monocyte surface antigen CD14 induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion.

Murine anti-CD14 mAb which recognize different CD14 epitopes induced marked homotypic adhesion of normal human monocytes. Induction of aggregation by anti-CD14 mAb required Mg2+, occurred at an optimal temperature of 37 degrees C, but not at 4 degrees C, and exhibited a kinetics which differed from adhesion triggered by IFN-gamma and anti-CD43 mAb. Monocyte adhesion induced by anti-CD14 mAb required neither Fcy gamma R engagement nor cross-linking of CD14, because adhesion was induced by F(ab)'2 fragments, as well as by monovalent F(ab) fragments of anti-CD14 mAb. mAb to CD11a, CD18, and intercellular adhesion molecule-1 (ICAM-1), but not antibodies to CD11b and CD11c, inhibited monocyte adhesion induced by CD14 engagement. These results indicate that CD14-dependent adhesion is mediated by lymphocyte function-associated Ag-1/ICAM-1 interactions. This was confirmed by the absence of aggregation in anti-CD14-stimulated cells from a patient with leukocyte adhesion deficiency. Monocyte adhesion upon CD14 engagement was blocked by an inhibitor of protein kinases, sphingosine. This suggests that protein kinases play a role in the intracellular signaling pathway(s) which couple CD14 to lymphocyte function-associated Ag-1/ICAM-1.