Lack of heparan sulfate expression in B-cell lines: implications for Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus 68 infections

Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS.     

In vitro and in vivo activation induces BAFF and APRIL expression in B cells

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.     

Heparan sulfate is an attachment factor for foamy virus entry

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.     

Dysregulated Cytokine Production by Dendritic Cells Modulates B Cell Responses in the NZM2410 Mouse Model of Lupus

The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-γ, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1⁺ cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1⁺ cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-γ production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.     

Heparan Sulfate Biosynthesis: Regulation and Variability

Nearly all vertebrate cells have been shown to express heparan sulfate proteoglycans (HSPGs) at the cell surface. The HSPGs bind to many secreted signaling proteins, including numerous growth factors, cytokines, and morphogens, to affect their tissue distribution and signaling. The heparan sulfate (HS) chains may have variable length and may differ with regard to both degree and pattern of sulfation. As the sulfation pattern of HS chains in most cases will determine if an interaction with a potential ligand will take place, as well as the affinity of the interaction, a key to understanding the function of HSPGs is to clarify how HS biosynthesis is regulated in different biological contexts. This review provides an introduction to the current understanding of HS biosynthesis and its regulation, and identifies research areas where more knowledge is needed to better understand how the HS biosynthetic machinery works.     

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a strategy that appears to be conserved between at least EBV and MHV68.     

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.     

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.     

Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain- specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.     

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.     

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.     

Heparan sulfate-independent infection attenuates high-neurovirulence GDVII virus-induced encephalitis

The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HS-deficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.     

Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity

HS (heparan sulfate) is essential for normal embryonic development. This requirement is due to the obligatory role for HS in the signalling pathways of many growth factors and morphogens that bind to sulfated domains in the HS polymer chain. The sulfation patterning of HS is determined by a complex interplay of Golgi-located N- and O-sulfotransferases which sulfate the heparan precursor and cell surface endosulfatases that selectively remove 6-O-sulfates from mature HS chains. In the present study we generated single or double knock-out mice for the two murine endosulfatases mSulf1 and mSulf2. Detailed structural analysis of HS from mSulf1-/- fibroblasts showed a striking increase in 6-O-sulfation, which was not seen in mSulf2-/- HS. Intriguingly, the level of 6-O-sulfation in the double mSulf1-/-/2-/- HS was significantly higher than that observed in the mSulf1-/- counterpart. These data imply that mSulf1 and mSulf2 are functionally co-operative. Unlike their avian orthologues, mammalian Sulf activities are not restricted to the highly sulfated S-domains of HS. Mitogenesis assays with FGF2 (fibroblast growth factor 2) revealed that Sulf activity decreases the activating potential of newly-synthesized HS, suggesting an important role for these enzymes in cell growth regulation in embryonic and adult tissues.     

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.     

Selection of COS cell mutants defective in the biosynthesis of heparan sulfate proteoglycan.

A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40-70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST) gene. Structure, expression, and function in the formation of the tracheal system

Heparan sulfate, one of the most abundant components of the cell surface and the extracellular matrix, is involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate chains have markedly heterogeneous structures in which distinct sequences of sulfate groups determine specific binding properties. Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and characterized Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST). The deduced primary structure of dHS6ST exhibited several common features found in those of mammalian HS6STs. We confirmed that, when the protein encoded by the cDNA was expressed in COS-7 cells, it showed HS6ST activity. Whole mount in situ hybridization revealed highly specific expression of dHS6ST mRNA in embryonic tracheal cells. The spatial and temporal pattern of dHS6ST expression in these cells clearly resembles that of the Drosophila fibroblast growth factor (FGF) receptor, breathless (btl). RNA interference experiments demonstrated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These phenotypes were reminiscent of the defects observed in mutants of FGF signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development in Drosophila and suggest the evolutionally conserved roles of 6-O-sulfated heparan sulfate in FGF signaling.     

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.     

Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin-like) activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation.