- Email: [email protected]
Ruminant cluster CD14
Veterinary
Vetetinarg immunology and immunopathology
Immunology and lmmunopathology 52 (1996) 245-248
Section 3
Ruminant cluster CD 14 P. Berthon a’*, J. Hopkins b a[NRA,
Luhomtoire
de Puthologie
Infectiruse
et Immunolo~re,
Nouzilly, b Deprtmmt
of’Veterinury
Pathology.
Uniurrsity
Equip
G&Gtiyur
et Immunite’, 37380
France
of’EdinhurKh,
Summerhull.
Edinburgh.
EH9 IQH,
UK
Abstract
Following the analysis of flow cytometry data on ovine and bovine cells and immunoprecipitation, immunohistochemistry and competitive binding studies, a cluster of three monoclonal antibodies (mAbs) (VPM65, VPM66 and VPM67) appeared to be specific for the ruminant CD14 (Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins, J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. Immunopathol. in press; Hopkins, J. and Gupta, V.K., 1996. Characterization of 3rd Worshop monoclonal antibodies specific for sheep macrophages/monocytes. Vet. Immunbl. Immunopathol., 52:329-339). According to flow cytometry results from human CDlCtransfected COS-7 cells and from bovine peripheral blood mononuclear cells, another mAb (CC-G33), not submitted to the Workshop, recognised a bovine CD14 epitope which is conserved in the human CD14 (Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol., 52: 323-328). Keywords;
CD1 4; LPS-receptor; Myeloid antigen; Ruminant; Leukocytes
1. Immunochemistry The three mAbs VPM6.5, VPM66 and VPM67 immunoprecipitated from sheep alveolar macrophage lysate a single band molecule with M, 55 000, under reducing and non-reducing conditions. Following deglycosylation with N-glycosidase F, the apparent
Abbreviations:
CD = Cluster of differentiation;
FGF = Fibroblast
growth
factor;
G-CSF = Granulocyte-
colony stimulating factor; GM-CSF = Granulocyte/macrophage-colony stimulating factor; GPI = Glycosyl phosphatidylinositol; IL = Interleukin; LPS = Lipopolysaccharide; mAb = Monoclonal antibody; PDGF = Platelet-derived growth factor; PI-PLC = Phosphatidylinositol phospholipase C; TGF-P = Transforming factor-p; TNF-a = Tumor necrosis factor-cu _ Corresponding author. Tel.: 33 47 42 78 68; fax: 33 47 42 77 79; e-mail: [email protected]. 0165-2427/96/$15.00 Published by Elsevier Science B.V. SO 165.2427(96)05568-7
PII
growth
246
P. Berthon,
J. Hopkins/
Veterinury
lmmunolo~y
and lnrmunr~purholo~y
52 (1996) 245-248
molecular weight of the antigen was reduced by M, 2000-3000. Phosphatidylinositol phospholipase C (PI-PLC) treatment of ovine alveolar macrophages and monocytes abolished the binding of these three mAbs to those cells, suggesting that the antigen recognised by these three mAbs is linked to the cell surface via a glycosyl phosphatidylinositol (GPI) anchor (Gupta et al., 1996; Hopkins and Gupta, 1996, this volume). These data are in agreement with those previously obtained from different animal species: the CD14 is a M, 5.5 000 glycoprotein, GPI-anchored in the cell membrane (Goyert et al., 1986; Wright et al., 1990). But the ovine CD14 molecule slightly differs from the human and bovine CD14 homologues with a 4-6% instead of 20% N-linked carbohydrate.
2. Cellular expression The mAb CC-G33 was first selected according to its specificity for bovine monocytes from peripheral blood mononuclear cells (Sopp et al., 1996, this volume). Data from flow cytometry analysis indicated that the three mAbs, VPM65, VPM66 and VPM67 showed identical characteristics of cellular labelling. They specifically reacted with sheep alveolar macrophages and peripheral blood mononuclear cells without staining peripheral blood lymphocytes. They strongly labelled ovine peripheral blood granulocytes and presented a slight reactivity for dendritic cells (Gupta et al., 1996). Similar patterns of labelling were observed on bovine leukocytes, except for granulocytes. Only one mAb, VPM65, was able to induce a signal on some bovine data from sheep neutrophils (Sopp et al., 1996, this volume). Immunohistochemical tissue sections revealed that these three mAbs bound to the macrophage cell subset in all tissue tested (Gupta et al., 1996). These mAbs were also able to stain some neutrophils and follicular dendritic cells (Berthon et al., 1996, this volume), as previously described in other species (Ziegler-Heitbrock and Ulevitch, 1993; Detmers et al., 1995; Lindhout and De Groot, 1995). The CD14 antigen appears to be also expressed on human B cells (Ziegler-Heitbrock et al., 1994) and human microglial cells (Peterson et al., 1995). According to their tissue localisation, macrophages show different intensities of labelling with anti-CD1 4 antibodies. Spleen macrophages, Klippfer cells, peritoneal and pleura1 macrophages appear to express high levels of CD14 antigen, while a low level is detected on alveolar macrophages. No CD14 is observed from early progenitor cells. Its expression increases with maturation towards monocytes and appears to be down-regulated by interleukin-4 (IL-4) (Ziegler-Heitbrock and Ulevitch, 1993).
3. Function The CD14 molecule mainly acts as a receptor for the lipopolysaccharide (LPS) (Wright et al., 1990) which is a major component of the cell wall of Gram negative bacteria and is responsible for the activation of myeloid cells (Wright et al., 1990; Tobias and Ulevitch, 1993; Schumann et al., 1994; Ulevitch and Tobias, 1994). The
P. Berthon.
J. Hopkins
/ Vrtrrrnury
Immunolopy
crnd Immuttcpuhoio~y
52 (19961245-248
241
CD14 antigen is also involved in the recognition of some cell wall components from Gram positive bacteria and Mycobacteria (Pugin et al., 1994). In monocytes, the CD1 4 activation pathway results in the release of pro-inflammatory cytokines such as IL- 1, IL-6, IL-S and tumor necrosis factor a (TNF-o) (Dentener et al.. 1993; Schumann et al., 1994). LPS-stimulated macrophages produce some polypeptides which induce cell growth, including fibroblast growth factor (FGF), transforming growth factor-p (TGF-l3) and platelet-derived growth factor (PDGF). In monocytic cells, LPS also induces the secretion of haematopoietic growth factors such as granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and IL-3 which are involved in the recruitment of leukocytes from the bone marrow. In vivo, this CD16dependent activation pathway of myeloid cells results in the development of an inflammatory response, a host defense mechanism against bacterial invasion that can also induce some tissue damages (Schumann et al., 1994). In addition, the CD14 antigen appears also to play a role in cell-cell interactions (Lauener et al., 1990; ZieglerHeitbrock and Ulevitch, 1993: Schumann et al.. 1994).
4. Human homologue Homology with the human CD14 antigen is based on the M, of the immunoprecipitated glycoprotein and on the cellular and tissue distribution. In addition, the mAb CC-G33, initially raised against the bovine CD14, cross-reacts with the human CD14 antigen expressed on transfected COS-7 cells and recognises a bovine CD14 epitope identical or closely related to the one defined by the anti-human CD14 mAb S39 (Yang et al., 1995; Sopp et al., 1996, this volume). Results from antigen-specific capture ELBA showed that the anti-human CD 14 mAb TcK4 (Gadd, 1989) reacted specifically with the ovine alveolar macrophage antigen captured by VPM6.5, indicating that these two mAbs recognise different epitopes of the ovine CD14 (Jacobsen et al., 1993; Gupta et al., 1996; Hopkins and Gupta, 1996, this volume). Data obtained by competitive binding on ovine alveolar macrophages showed that the three mAbs, VPM65, VPM66 and VPM67, seemed to be specific for the same or very closely related epitopes of the antigen (Gupta et al., 1996). The analysis of bivariate plots obtained with a flow cytometry two-colour staining method on bovine peripheral blood mononuclear cells suggests that CC-G33 in one hand and TUK4, VPM65, VPM66 and VPM67 in the other hand recognise different epitopes of the same molecule (Sopp et al., 1996, this volume).
References Berthon, P., Bernard, F., Olivier, M., Bernard, S. and Lantier, F., 1996. Immunohistochemical study on the reactivity of Workshop monoclonal antibodies with sheep lymph nodes. Vet. Immunol. Immunopathol. 52: 393-401. Dentener, M.A., Bazil, V., Von, Asmuth, E.J.U., C&a, M. and Buurman, W.A., 1993. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-u, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J. Immunol., 150: 28852891.
248
P. Berthon,
J. Hopkins/
Veterinary
Immunology
crnd Immunoputhology
52 (1996) 245-248
Detmers, P.A., Thou, D., Powell, D., Lichenstein, H., Kelley, M. and Pironkova, R., 1995. Endotoxin receptors (CD141 are found with CD16 (FcyRIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase. J. Immunol., 155: 2085-2095. Gadd, S., 1989. In: Cluster report: CD14 Knapp W. Leucocyte Typin, 0 IV: white Cell Differentiation Antigens. Oxford University Press, Oxford. Goyert, S.M., Ferrero, E.M., Seremetis, S.V., Winchester, R.J., Silver, J. and Mattison, A.C., 1986. Biochemistry and expression of myelomonocytic antigens. J. Immunol., 137: 3909-3914. Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins, J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. Immunopathol., in press. Hopkins, J. and Gupta, V.K., 1996. Characterization of 3rd Worshop monoclonal antibodies specific for sheep macrophages/monocytes. Vet. Immunol. Immunopathol., in press. Jacobsen, C.N., Aasted, B., Broe, M.C. and Petersen, J.L., 1993. Reactivities of 20 anti-human monoclonal antibodies with leukocytes from ten different animal species. Vet. Immunol. Immunopathol., 39: 461-466. Lauener, R.P., Geha, R.S. and Vercelli, D., 1990. Engagement of the monocyte surface antigen CD14 induces lymphocyte function-associated antigen- I /intercellular adhesion molecule- I dependent homotypic adhesion. J. Immunol., 145: 1390- 1394. Lindhout, E. and De Groot, C., 1995. Follicular dendritic cells and apoptosis: life and death in the germinal centre. Histochem. J., 27: 167-183. Peterson, P.K., Gekker, G., Hu, S., Sheng, W.S., Anderson, W.R., Ulevitch, R.J., Tobias, P.S., Gustafson, K.V., Molitor, T.W. and Chao, C.C., 1995. CD14 receptor-mediated uptake of nonopsonized Mycohuctrrium tuherculosi.s by human microglia. Infect. Immun., 63: 159% 1602. Pugin, J., Heumann, D., Tomasz, A., Kravchenko, V.V., Akamatsu, Y., Nishijima, M., Glauser, M.P., Tobias, P.S. and Ulevitch, R.J., 1994. D14 is a pattern recognition receptor Immunity. I: 509-516. Schumann R.R., Rietschel E.T. and oppnow, H., 1994. The role of CD14 and lipopolysaccharide-binding protein (LBP) in the activation of different cell types by endotoxin. Med. Microbial. Immunol., 183: 279-297. Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol. 52: 323-328. Tobias, P.S. and Ulevitch, R.J., 1993. Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation. Immunobiology, 187: 227-232. Ulevitch, R.J. and Tobias, P.S., 1994. Recognition of endotoxin by cells leading to transmembrane signaling. Curr. Opin. Immunol., 6: 125-130. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J. and Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science, 249: I43 I - 1433. Yang, Z., Carter, C.D., Miller, M.S. and Bochsler, P.N., 1995. CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro. Infect. Immun., 63: 5 I-56. Ziegler-Heitbrock, H.W.L. and Ulevitch, R.J., 1993. CDl4: Cell surface receptor and differentiation marker. Immunol. Today, 14: 121-125. Ziegler-Heitbrock, H.W.L., Pechumer, H., Petersmann, I., Durieux, J.J., Vita, N., Labeta, M.O. and Striibel, M., 1994. CD14 is expressed and functional in human B cells. Eur. J. Immunol., 24: 1937-1940.
Vetetinarg immunology and immunopathology
Immunology and lmmunopathology 52 (1996) 245-248
Section 3
Ruminant cluster CD 14 P. Berthon a’*, J. Hopkins b a[NRA,
Luhomtoire
de Puthologie
Infectiruse
et Immunolo~re,
Nouzilly, b Deprtmmt
of’Veterinury
Pathology.
Uniurrsity
Equip
G&Gtiyur
et Immunite’, 37380
France
of’EdinhurKh,
Summerhull.
Edinburgh.
EH9 IQH,
UK
Abstract
Following the analysis of flow cytometry data on ovine and bovine cells and immunoprecipitation, immunohistochemistry and competitive binding studies, a cluster of three monoclonal antibodies (mAbs) (VPM65, VPM66 and VPM67) appeared to be specific for the ruminant CD14 (Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins, J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. Immunopathol. in press; Hopkins, J. and Gupta, V.K., 1996. Characterization of 3rd Worshop monoclonal antibodies specific for sheep macrophages/monocytes. Vet. Immunbl. Immunopathol., 52:329-339). According to flow cytometry results from human CDlCtransfected COS-7 cells and from bovine peripheral blood mononuclear cells, another mAb (CC-G33), not submitted to the Workshop, recognised a bovine CD14 epitope which is conserved in the human CD14 (Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol., 52: 323-328). Keywords;
CD1 4; LPS-receptor; Myeloid antigen; Ruminant; Leukocytes
1. Immunochemistry The three mAbs VPM6.5, VPM66 and VPM67 immunoprecipitated from sheep alveolar macrophage lysate a single band molecule with M, 55 000, under reducing and non-reducing conditions. Following deglycosylation with N-glycosidase F, the apparent
Abbreviations:
CD = Cluster of differentiation;
FGF = Fibroblast
growth
factor;
G-CSF = Granulocyte-
colony stimulating factor; GM-CSF = Granulocyte/macrophage-colony stimulating factor; GPI = Glycosyl phosphatidylinositol; IL = Interleukin; LPS = Lipopolysaccharide; mAb = Monoclonal antibody; PDGF = Platelet-derived growth factor; PI-PLC = Phosphatidylinositol phospholipase C; TGF-P = Transforming factor-p; TNF-a = Tumor necrosis factor-cu _ Corresponding author. Tel.: 33 47 42 78 68; fax: 33 47 42 77 79; e-mail: [email protected]. 0165-2427/96/$15.00 Published by Elsevier Science B.V. SO 165.2427(96)05568-7
PII
growth
246
P. Berthon,
J. Hopkins/
Veterinury
lmmunolo~y
and lnrmunr~purholo~y
52 (1996) 245-248
molecular weight of the antigen was reduced by M, 2000-3000. Phosphatidylinositol phospholipase C (PI-PLC) treatment of ovine alveolar macrophages and monocytes abolished the binding of these three mAbs to those cells, suggesting that the antigen recognised by these three mAbs is linked to the cell surface via a glycosyl phosphatidylinositol (GPI) anchor (Gupta et al., 1996; Hopkins and Gupta, 1996, this volume). These data are in agreement with those previously obtained from different animal species: the CD14 is a M, 5.5 000 glycoprotein, GPI-anchored in the cell membrane (Goyert et al., 1986; Wright et al., 1990). But the ovine CD14 molecule slightly differs from the human and bovine CD14 homologues with a 4-6% instead of 20% N-linked carbohydrate.
2. Cellular expression The mAb CC-G33 was first selected according to its specificity for bovine monocytes from peripheral blood mononuclear cells (Sopp et al., 1996, this volume). Data from flow cytometry analysis indicated that the three mAbs, VPM65, VPM66 and VPM67 showed identical characteristics of cellular labelling. They specifically reacted with sheep alveolar macrophages and peripheral blood mononuclear cells without staining peripheral blood lymphocytes. They strongly labelled ovine peripheral blood granulocytes and presented a slight reactivity for dendritic cells (Gupta et al., 1996). Similar patterns of labelling were observed on bovine leukocytes, except for granulocytes. Only one mAb, VPM65, was able to induce a signal on some bovine data from sheep neutrophils (Sopp et al., 1996, this volume). Immunohistochemical tissue sections revealed that these three mAbs bound to the macrophage cell subset in all tissue tested (Gupta et al., 1996). These mAbs were also able to stain some neutrophils and follicular dendritic cells (Berthon et al., 1996, this volume), as previously described in other species (Ziegler-Heitbrock and Ulevitch, 1993; Detmers et al., 1995; Lindhout and De Groot, 1995). The CD14 antigen appears to be also expressed on human B cells (Ziegler-Heitbrock et al., 1994) and human microglial cells (Peterson et al., 1995). According to their tissue localisation, macrophages show different intensities of labelling with anti-CD1 4 antibodies. Spleen macrophages, Klippfer cells, peritoneal and pleura1 macrophages appear to express high levels of CD14 antigen, while a low level is detected on alveolar macrophages. No CD14 is observed from early progenitor cells. Its expression increases with maturation towards monocytes and appears to be down-regulated by interleukin-4 (IL-4) (Ziegler-Heitbrock and Ulevitch, 1993).
3. Function The CD14 molecule mainly acts as a receptor for the lipopolysaccharide (LPS) (Wright et al., 1990) which is a major component of the cell wall of Gram negative bacteria and is responsible for the activation of myeloid cells (Wright et al., 1990; Tobias and Ulevitch, 1993; Schumann et al., 1994; Ulevitch and Tobias, 1994). The
P. Berthon.
J. Hopkins
/ Vrtrrrnury
Immunolopy
crnd Immuttcpuhoio~y
52 (19961245-248
241
CD14 antigen is also involved in the recognition of some cell wall components from Gram positive bacteria and Mycobacteria (Pugin et al., 1994). In monocytes, the CD1 4 activation pathway results in the release of pro-inflammatory cytokines such as IL- 1, IL-6, IL-S and tumor necrosis factor a (TNF-o) (Dentener et al.. 1993; Schumann et al., 1994). LPS-stimulated macrophages produce some polypeptides which induce cell growth, including fibroblast growth factor (FGF), transforming growth factor-p (TGF-l3) and platelet-derived growth factor (PDGF). In monocytic cells, LPS also induces the secretion of haematopoietic growth factors such as granulocyte-colony stimulating factor (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and IL-3 which are involved in the recruitment of leukocytes from the bone marrow. In vivo, this CD16dependent activation pathway of myeloid cells results in the development of an inflammatory response, a host defense mechanism against bacterial invasion that can also induce some tissue damages (Schumann et al., 1994). In addition, the CD14 antigen appears also to play a role in cell-cell interactions (Lauener et al., 1990; ZieglerHeitbrock and Ulevitch, 1993: Schumann et al.. 1994).
4. Human homologue Homology with the human CD14 antigen is based on the M, of the immunoprecipitated glycoprotein and on the cellular and tissue distribution. In addition, the mAb CC-G33, initially raised against the bovine CD14, cross-reacts with the human CD14 antigen expressed on transfected COS-7 cells and recognises a bovine CD14 epitope identical or closely related to the one defined by the anti-human CD14 mAb S39 (Yang et al., 1995; Sopp et al., 1996, this volume). Results from antigen-specific capture ELBA showed that the anti-human CD 14 mAb TcK4 (Gadd, 1989) reacted specifically with the ovine alveolar macrophage antigen captured by VPM6.5, indicating that these two mAbs recognise different epitopes of the ovine CD14 (Jacobsen et al., 1993; Gupta et al., 1996; Hopkins and Gupta, 1996, this volume). Data obtained by competitive binding on ovine alveolar macrophages showed that the three mAbs, VPM65, VPM66 and VPM67, seemed to be specific for the same or very closely related epitopes of the antigen (Gupta et al., 1996). The analysis of bivariate plots obtained with a flow cytometry two-colour staining method on bovine peripheral blood mononuclear cells suggests that CC-G33 in one hand and TUK4, VPM65, VPM66 and VPM67 in the other hand recognise different epitopes of the same molecule (Sopp et al., 1996, this volume).
References Berthon, P., Bernard, F., Olivier, M., Bernard, S. and Lantier, F., 1996. Immunohistochemical study on the reactivity of Workshop monoclonal antibodies with sheep lymph nodes. Vet. Immunol. Immunopathol. 52: 393-401. Dentener, M.A., Bazil, V., Von, Asmuth, E.J.U., C&a, M. and Buurman, W.A., 1993. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-u, IL-6 and IL-8 release by human monocytes and alveolar macrophages. J. Immunol., 150: 28852891.
248
P. Berthon,
J. Hopkins/
Veterinary
Immunology
crnd Immunoputhology
52 (1996) 245-248
Detmers, P.A., Thou, D., Powell, D., Lichenstein, H., Kelley, M. and Pironkova, R., 1995. Endotoxin receptors (CD141 are found with CD16 (FcyRIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase. J. Immunol., 155: 2085-2095. Gadd, S., 1989. In: Cluster report: CD14 Knapp W. Leucocyte Typin, 0 IV: white Cell Differentiation Antigens. Oxford University Press, Oxford. Goyert, S.M., Ferrero, E.M., Seremetis, S.V., Winchester, R.J., Silver, J. and Mattison, A.C., 1986. Biochemistry and expression of myelomonocytic antigens. J. Immunol., 137: 3909-3914. Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins, J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. Immunopathol., in press. Hopkins, J. and Gupta, V.K., 1996. Characterization of 3rd Worshop monoclonal antibodies specific for sheep macrophages/monocytes. Vet. Immunol. Immunopathol., in press. Jacobsen, C.N., Aasted, B., Broe, M.C. and Petersen, J.L., 1993. Reactivities of 20 anti-human monoclonal antibodies with leukocytes from ten different animal species. Vet. Immunol. Immunopathol., 39: 461-466. Lauener, R.P., Geha, R.S. and Vercelli, D., 1990. Engagement of the monocyte surface antigen CD14 induces lymphocyte function-associated antigen- I /intercellular adhesion molecule- I dependent homotypic adhesion. J. Immunol., 145: 1390- 1394. Lindhout, E. and De Groot, C., 1995. Follicular dendritic cells and apoptosis: life and death in the germinal centre. Histochem. J., 27: 167-183. Peterson, P.K., Gekker, G., Hu, S., Sheng, W.S., Anderson, W.R., Ulevitch, R.J., Tobias, P.S., Gustafson, K.V., Molitor, T.W. and Chao, C.C., 1995. CD14 receptor-mediated uptake of nonopsonized Mycohuctrrium tuherculosi.s by human microglia. Infect. Immun., 63: 159% 1602. Pugin, J., Heumann, D., Tomasz, A., Kravchenko, V.V., Akamatsu, Y., Nishijima, M., Glauser, M.P., Tobias, P.S. and Ulevitch, R.J., 1994. D14 is a pattern recognition receptor Immunity. I: 509-516. Schumann R.R., Rietschel E.T. and oppnow, H., 1994. The role of CD14 and lipopolysaccharide-binding protein (LBP) in the activation of different cell types by endotoxin. Med. Microbial. Immunol., 183: 279-297. Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol. 52: 323-328. Tobias, P.S. and Ulevitch, R.J., 1993. Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation. Immunobiology, 187: 227-232. Ulevitch, R.J. and Tobias, P.S., 1994. Recognition of endotoxin by cells leading to transmembrane signaling. Curr. Opin. Immunol., 6: 125-130. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J. and Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science, 249: I43 I - 1433. Yang, Z., Carter, C.D., Miller, M.S. and Bochsler, P.N., 1995. CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro. Infect. Immun., 63: 5 I-56. Ziegler-Heitbrock, H.W.L. and Ulevitch, R.J., 1993. CDl4: Cell surface receptor and differentiation marker. Immunol. Today, 14: 121-125. Ziegler-Heitbrock, H.W.L., Pechumer, H., Petersmann, I., Durieux, J.J., Vita, N., Labeta, M.O. and Striibel, M., 1994. CD14 is expressed and functional in human B cells. Eur. J. Immunol., 24: 1937-1940.