Vol. 166, No. 2, 1990 January 30, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 848-851
LEUKOTRIENEB4lNDUCEDHYPERADHESlVENESSOF ENDOTHELIAL CELLS FOR NEUTROPHIIS
Jan Palm&d, Peter Lin&tn%n and Richard Werner Department of Medicine 3, The Karolinska Institute at Wdersjukhuset, S-10064 Stockholm 60, Sweden
Received
December
4, 1989
Leukotriene B4 (LTB4) induced a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HLJVEC), leading to increased binding of neutrophil granulocytes (PMN). The effect of LTM was more rapidly emerging and transient than responses to platelet activating factor (PAF), thrombin and phorbol my&t&e acetate (PMA). At O&M of LTB4, it was comparable to hyperadhesiveness induced by 1 U/ml of thrombin, but less than that conferred by 0.1 pM of PAF and PMA. The adherence response to LTB4 was specific since the structural analogue 5$12S-diiETE, which lacks PMN-stimulating effects, failed to promote HUVEC adhesiveness. o 1990 Academic Press, Inc.
Endotehelial
cells participate actively in the emigration of PMN granulocytes by providing adhesive
structures. The expression of these structures can be upregulated mediators. One mediator
by means of inflammatory
class confers a rapid hyperadhesiveness, dependent
on endogenous
formation of platelet activating factor (PAF). To this class belongs thrombin and leukotriene C4 (l5). A more slowly emerging hyperadhesiveness has been observed after treating endothelial with interleukin
cells
1, tumor necrosis factor and gamma interferon.
We describe here that leukotriene
B4 (LTB4)
induces a rapidly emerging and vanishing
hyperadhesive state in cultured human umbilical vein endothelial PAF, phorbol my&ate
cells (HUVEC).
Responses to
acetate (PMA) and thrombin appeared later and persisted longer.
kinetics of these LTB4 and PAF responses resemble the kinetics of the hyperpolarization
The
changes
these agents induce in HUVEC (6) and a number of functional responses in neutro- and eosinophil granulocytes (7-9). MATERIAL
AND
METHODS
Ch~miculs: Human serum albumin, PMA, PAF, heparin, gelatin and thrombin were obtained from Sigma Chemicals Co. (St. Louis, MO), leukotriene B4 (LTB4), dissolved in ethanol and
Abbreviations: HBSS, Hank’s balanced salt solution; HSA, human serum albumin; HUVEC, human umbiIica1 vein endothelial cefls; LTB4, leukotriene B4 (5(S),l2(R)dihydroxy-6,8,10,14eicosatetraenoic acid); SS,12%IiHETE, S(S),12(S)-dihydroxy-6-trans,8-cis,lO-trans,I4-cis-eicosatetraenoic acid; PAF, platelet activating factor (lQhexadecy1, 2aceXyl-phosphatidyk&o~in; PMA, phorbol myrime acetate; PMN, polymorphonuclear neutrophil granulocyte. 0006-291X/90 Copyright Ali rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any formreserved.
848
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
SS,12S-diHETE were kind gifts from Drs. J. Rokach (Merck-Frosst Inc., Dorval, Canada). Hanks balanced salt solution (HBSS) was from Natl. Bact. Lab. (Stockholm). ECGS was from Collaborative Research Inc. (Bedford, MA) and RPM1 1640, fetal calf serum, penicillin and streptomycin from Gibco (Paisley, Scotland) Polystyrene tissuse culture dishes, 16 mm, were from Nunc (Roskilde, Denmark), Percoll was from Pharmacia (Uppsala, Sweden) and EDTA from Merck (Darmstadt, FRG). Culture of HWEC: Endothelial cells were obtained from human umbilical veins by treatment with 0.2% collagenase as described (6,lO). Cells were suspended in culture medium (RPM1 1640 with 20% fetal calf serum, 90 g/ml of heparin, 50 pg endothelial cell growth factor, 100 U penicillin/ml and 100 mg streptomycin/ml) and grown in SO cm2 tissue culture flasks precoated with 2% gelatin. Culture medium was changed the next day and then twice weekly. HUVEC were ttypsinized when confluent, resuspended in medium and either seeded into new culture flasks (for a maximum of 2 passages, once per week) or seeded into 16 mm culture wells. When those new monolayers were established in the culture wells, they were used for adherence assessments. Culture medium was changed the day before adherence tests. By using an indirect immunofluorescence staining for factor VIII related antigen monolayers were identified as homogenous HUVEC layers. Neutrophils and the adherence assay: PMNs were obtained from healthy donors by a discontinous Percoll gradient centrifugation (9). The purified PMNs (>95% purity and viability) were resuspended in HBSS with 0.4% HSA Adherence to HUVEC monolayers was assessed as described (11). Briefly, monolayers were treated with LTB4 or indicated stimuli at 37°C. After 2 washes (which has been shown to remove 99.2% of radiolabelled LTB+ Lindstrom et al, to be published), PMNs (0.5 ml with appr. 2.5~10~ cells/ml, warmed at 37°C for 10 min) were added. After careful mixing the dish was incubated at 37°C and 5% CO,. At the end of the indicated incubation period the fluids were aspirated and added to 0.1 M EDTA containing saline (to minimize aggregation and further adhesion of PMNs). The wells were then washed twice with 250 ul HBSS with 0.4% HSA The number of PMNs in the combined aspirates and washing fluids were subsequently counted in a cell counter and adherence was, after correction for dilution, calculated from the formula: % adherence=(l-nonadherent cells/added cells) x 100. The increment of adherence (hyperadherence) induced by stimuli is given by subtracting the value for the HBSS treated PMNs from that of stimulated cells. Stained smears showed that only PMNs were counted and that no disruption of HUVEC monolayers had occurred. Spontaneous adherence of PMNs remained constant from 5 to 30 min at 11.1+0.6, n=39 (mean and SEM). RESULTS HUVEC
monolayers treated with LTB4 at the optimal concentration
for chemotaxis of PMNs,
100 nM, bound twice as many PMNs as HBSS treated control cells (Fig. 1). The kinetics of the response was of significance for detection of the LTB4 effect. LTB4 treatment of HUVEC for 5 min was found to be optimal for induction of HUVEC hyperadhesiveness. When prolonged for more than 15 min no hyperadhesiveness was noted. Similarly, the subsequent incubation neutrophils
period with
showed highest adherence values when run for 5 min, Only half of that number of
PMNs were bound to HUVEC when the PMN incubation was 15 min. No increments above HBSS treated controls were noted when the adherence phase was prolonged to 30 min. HUVEC treated with 1 U thrombin/ml or 100 nM of PAF and PMA exhibited hyperadhesiveness for PMNs, but those responses became maximal later than for LTB4 with regard to both treatment and PMN adherence periods (Fig. 1). PMA conferred the slowest onset. Thrombin
was associated
with a magnitude of hyperadhesion similar to that of LTB4, whereas maximal responses to PMA and PAF were 2.6 and 2.1-fold higher, respectively. The specificity of the LTB4 response was assessed by treating HUVEC monolayers with 5S,12SdiHETE,
a strucural analogue
to LTB4, without
effects on PMN oxidative metabolism
or
chemotaxis (12). As shown in Table 1, this lipoxygenase product conferred no hyperadhesiveness of HUVEC. 849
Vol.
166, No. 2, 1990
BIOCHEMICAL
Treatment
5
of HUVEC
AND BIOPHYSICAL
for 5 min
Treatment
0
30
15
minutes
of PMN
RESEARCH COMMUNICATIONS
of HUVEC
5
for 15 min
15
30
adherence
Figure 1. HUVEC hyperadhesiveness induced by LTB4, PAP and PMA (all at 100 nM) and thrombll (at 1 U/ml). HUVEC were treated for 5 (left panel) or 15 min (right panel) with the stimuli and then washed. Subsequently, PMNs were added and adherence of PMNs were assessed after incubation for 5,15 or 30 min at 37°C. Data represent mean and SE values for 3-4 separate experiments, run in du- or triplicates. All values are given as net increase of PMN binding above HBSS stimulated cells, i.e. hyperadhesiveness.
DISCUSSION This study has demonstrated hyperadhesive state of HUVEC analoque SS,12S-diHETE,
that LTB4
is able to induce a rapidly occurring
but transient
monolayers. The effect of LTB4 is specific, since the structural
which differs from LTB4 with regard to cis-trans configurations and has
no neutrophil activating effect (12), was ineffective in the here described system. In previous studies LTB4 has been reported either to lack or to possess an adherence promoting activity on endothelial
cells (4,13). As shown here, the transient nature of the LTB4 response
required careful evaluation of early time points to be detected. In contrast, responses to thrombin and PAF, and particularly to PMA, emerged slowly and were protracted. The maximal responses to equimolar concentrations of PAF and PM.4 were stronger than those to LTB4 (and thrombin). Some of these response characteristics have been reported
previously but no comparisons have
appeared.
Table
1.
PMN hypemdhesiveness
HUVEC treated with
% hyperadherence
to HVVEC
n
LTB4, loo nM
+10.3*1.3
17
SS,12SdiHETE, 100 nM
-1.7f2.0
3
Mean and SE values for hyperadherenm of PMNs (i.e. the increase of adherence above HBSStreated controls). HUVEC monolayers were treated with the stimuli for 5 mitt, washed, and PMNs were added. Hyperadherence was assessed5 min later. n=number of separate experiments, run in dupli- or triplicates. The negative value for SSJ2SdiHETE denotes that adherence values were lower than for HBSS stimulated controls.
850
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The rapid kinetics of the LTB4 adhesion response is similar to what we have observed LTB4 induced change of the membrane potential of HUVEC concentration
(6) as well as the cytosolic calcium
changes, aggregation and superoxide ion production
(7-9). This unique property of LTB4
for the
of neutrophils
and eosinophils
is probably expressed at a step of the stimulus response
coupling that is close to the surface receptor since the LTB4 induced generation of diacylglycerols and inositol phosphates exhibit similar kinetics (14,lS). It is not known whether HUVEC
express
surface receptors for LTBI ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (19X-05991,19P7fl95), The Swedish Association against Rheumatism, King Gustaf v’s SO- year Fund, the Funds of the Karolinska Institute, Sodersjukhuset, P&A Hedlund, S & E-C Hagberg, The Swedish Medical Society and Wrenade Liv Insurance Comp. The skilful technical assistance of Mrs. S. Myrin, P. Spingberg and Mr. C. Forsbom is gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Zimmerman, GA., Whatley, R.E., McIntyre, T.M. and Prescott, SM. (1987) Am. Rev. Resp. Dis. 136,204-207. Webster, R.0, Wysolmerski, R.B. and Lagunoff,D. (1986) Am. J. Pathol. 125369-378. Schleimer, R.P. and Rutledge B.K (1986) J. Immunol. 136,649-654. McIntyre, T.M., Zimmerman, GA and Prescott, SM. (1986) Proc. Natl. Acad. Sci. USA 83, 2204-2208. Garcia, J.G.N., Azghani, A, Callahan, KS. and Johnson, A.R. (1988) Thrombosis Res. 51, 83-96. Werner, R., Lindstrom, P. and Palmblad, J. (19SS) Biochem. Biophys. Res. Commun. 153, 805-810. Palmblad, J., Gyllenhammar, H., Lindgren, J.A. & Malmsten, C.L. (1984) J. Immunol. 132, 3041-3045. Palmblad, J., Gyllenhammar, H., Ringertz, B., Nilsson, E. and Cottell, B. (1988) Biochim. Biophys. Acta 970,92-102. Ringer@ B., Palmblad, J. and Lindgren, J.k (1985) J. Lab. Clin. Med. 106,132-140. Jaffe, EA., Nachman, R.L. Becker, C.G. and Miniek, R. (1973) J. Clin. Invest. 52,2745-2756. Lindstrom, P. and Palmblad, J. (1988) J. Clin. Lab. Immunol. 25,77-81. Palmblad, J, Uden, A-M., Lindgren, J.A., R&dmark, O., Hanson G. and Malmsten, C.L. (1982) FEBS Lett. 144,Sl-83. Hoover, R.L., Kamovsky, M.J., Austen, RF., Corey, E,J, and Lewis, R.A. (1984) Proc. Natl. Acad. Sci. USA 81,2191-2193. Riebman, J., Korchak, H.M., Vosshall, L.B., Haines, KA., Rich, AM. and Weissmann, G. (1988) J. Biol. Chem. 263,6322632X Truett, AP. III, Verghese, M.W., Dillon, S.B. and Snyderman, R. (1988) Proc. Natl. Acad. Sci. USA S5,1549-1553.
851
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 848-851
LEUKOTRIENEB4lNDUCEDHYPERADHESlVENESSOF ENDOTHELIAL CELLS FOR NEUTROPHIIS
Jan Palm&d, Peter Lin&tn%n and Richard Werner Department of Medicine 3, The Karolinska Institute at Wdersjukhuset, S-10064 Stockholm 60, Sweden
Received
December
4, 1989
Leukotriene B4 (LTB4) induced a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HLJVEC), leading to increased binding of neutrophil granulocytes (PMN). The effect of LTM was more rapidly emerging and transient than responses to platelet activating factor (PAF), thrombin and phorbol my&t&e acetate (PMA). At O&M of LTB4, it was comparable to hyperadhesiveness induced by 1 U/ml of thrombin, but less than that conferred by 0.1 pM of PAF and PMA. The adherence response to LTB4 was specific since the structural analogue 5$12S-diiETE, which lacks PMN-stimulating effects, failed to promote HUVEC adhesiveness. o 1990 Academic Press, Inc.
Endotehelial
cells participate actively in the emigration of PMN granulocytes by providing adhesive
structures. The expression of these structures can be upregulated mediators. One mediator
by means of inflammatory
class confers a rapid hyperadhesiveness, dependent
on endogenous
formation of platelet activating factor (PAF). To this class belongs thrombin and leukotriene C4 (l5). A more slowly emerging hyperadhesiveness has been observed after treating endothelial with interleukin
cells
1, tumor necrosis factor and gamma interferon.
We describe here that leukotriene
B4 (LTB4)
induces a rapidly emerging and vanishing
hyperadhesive state in cultured human umbilical vein endothelial PAF, phorbol my&ate
cells (HUVEC).
Responses to
acetate (PMA) and thrombin appeared later and persisted longer.
kinetics of these LTB4 and PAF responses resemble the kinetics of the hyperpolarization
The
changes
these agents induce in HUVEC (6) and a number of functional responses in neutro- and eosinophil granulocytes (7-9). MATERIAL
AND
METHODS
Ch~miculs: Human serum albumin, PMA, PAF, heparin, gelatin and thrombin were obtained from Sigma Chemicals Co. (St. Louis, MO), leukotriene B4 (LTB4), dissolved in ethanol and
Abbreviations: HBSS, Hank’s balanced salt solution; HSA, human serum albumin; HUVEC, human umbiIica1 vein endothelial cefls; LTB4, leukotriene B4 (5(S),l2(R)dihydroxy-6,8,10,14eicosatetraenoic acid); SS,12%IiHETE, S(S),12(S)-dihydroxy-6-trans,8-cis,lO-trans,I4-cis-eicosatetraenoic acid; PAF, platelet activating factor (lQhexadecy1, 2aceXyl-phosphatidyk&o~in; PMA, phorbol myrime acetate; PMN, polymorphonuclear neutrophil granulocyte. 0006-291X/90 Copyright Ali rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any formreserved.
848
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
SS,12S-diHETE were kind gifts from Drs. J. Rokach (Merck-Frosst Inc., Dorval, Canada). Hanks balanced salt solution (HBSS) was from Natl. Bact. Lab. (Stockholm). ECGS was from Collaborative Research Inc. (Bedford, MA) and RPM1 1640, fetal calf serum, penicillin and streptomycin from Gibco (Paisley, Scotland) Polystyrene tissuse culture dishes, 16 mm, were from Nunc (Roskilde, Denmark), Percoll was from Pharmacia (Uppsala, Sweden) and EDTA from Merck (Darmstadt, FRG). Culture of HWEC: Endothelial cells were obtained from human umbilical veins by treatment with 0.2% collagenase as described (6,lO). Cells were suspended in culture medium (RPM1 1640 with 20% fetal calf serum, 90 g/ml of heparin, 50 pg endothelial cell growth factor, 100 U penicillin/ml and 100 mg streptomycin/ml) and grown in SO cm2 tissue culture flasks precoated with 2% gelatin. Culture medium was changed the next day and then twice weekly. HUVEC were ttypsinized when confluent, resuspended in medium and either seeded into new culture flasks (for a maximum of 2 passages, once per week) or seeded into 16 mm culture wells. When those new monolayers were established in the culture wells, they were used for adherence assessments. Culture medium was changed the day before adherence tests. By using an indirect immunofluorescence staining for factor VIII related antigen monolayers were identified as homogenous HUVEC layers. Neutrophils and the adherence assay: PMNs were obtained from healthy donors by a discontinous Percoll gradient centrifugation (9). The purified PMNs (>95% purity and viability) were resuspended in HBSS with 0.4% HSA Adherence to HUVEC monolayers was assessed as described (11). Briefly, monolayers were treated with LTB4 or indicated stimuli at 37°C. After 2 washes (which has been shown to remove 99.2% of radiolabelled LTB+ Lindstrom et al, to be published), PMNs (0.5 ml with appr. 2.5~10~ cells/ml, warmed at 37°C for 10 min) were added. After careful mixing the dish was incubated at 37°C and 5% CO,. At the end of the indicated incubation period the fluids were aspirated and added to 0.1 M EDTA containing saline (to minimize aggregation and further adhesion of PMNs). The wells were then washed twice with 250 ul HBSS with 0.4% HSA The number of PMNs in the combined aspirates and washing fluids were subsequently counted in a cell counter and adherence was, after correction for dilution, calculated from the formula: % adherence=(l-nonadherent cells/added cells) x 100. The increment of adherence (hyperadherence) induced by stimuli is given by subtracting the value for the HBSS treated PMNs from that of stimulated cells. Stained smears showed that only PMNs were counted and that no disruption of HUVEC monolayers had occurred. Spontaneous adherence of PMNs remained constant from 5 to 30 min at 11.1+0.6, n=39 (mean and SEM). RESULTS HUVEC
monolayers treated with LTB4 at the optimal concentration
for chemotaxis of PMNs,
100 nM, bound twice as many PMNs as HBSS treated control cells (Fig. 1). The kinetics of the response was of significance for detection of the LTB4 effect. LTB4 treatment of HUVEC for 5 min was found to be optimal for induction of HUVEC hyperadhesiveness. When prolonged for more than 15 min no hyperadhesiveness was noted. Similarly, the subsequent incubation neutrophils
period with
showed highest adherence values when run for 5 min, Only half of that number of
PMNs were bound to HUVEC when the PMN incubation was 15 min. No increments above HBSS treated controls were noted when the adherence phase was prolonged to 30 min. HUVEC treated with 1 U thrombin/ml or 100 nM of PAF and PMA exhibited hyperadhesiveness for PMNs, but those responses became maximal later than for LTB4 with regard to both treatment and PMN adherence periods (Fig. 1). PMA conferred the slowest onset. Thrombin
was associated
with a magnitude of hyperadhesion similar to that of LTB4, whereas maximal responses to PMA and PAF were 2.6 and 2.1-fold higher, respectively. The specificity of the LTB4 response was assessed by treating HUVEC monolayers with 5S,12SdiHETE,
a strucural analogue
to LTB4, without
effects on PMN oxidative metabolism
or
chemotaxis (12). As shown in Table 1, this lipoxygenase product conferred no hyperadhesiveness of HUVEC. 849
Vol.
166, No. 2, 1990
BIOCHEMICAL
Treatment
5
of HUVEC
AND BIOPHYSICAL
for 5 min
Treatment
0
30
15
minutes
of PMN
RESEARCH COMMUNICATIONS
of HUVEC
5
for 15 min
15
30
adherence
Figure 1. HUVEC hyperadhesiveness induced by LTB4, PAP and PMA (all at 100 nM) and thrombll (at 1 U/ml). HUVEC were treated for 5 (left panel) or 15 min (right panel) with the stimuli and then washed. Subsequently, PMNs were added and adherence of PMNs were assessed after incubation for 5,15 or 30 min at 37°C. Data represent mean and SE values for 3-4 separate experiments, run in du- or triplicates. All values are given as net increase of PMN binding above HBSS stimulated cells, i.e. hyperadhesiveness.
DISCUSSION This study has demonstrated hyperadhesive state of HUVEC analoque SS,12S-diHETE,
that LTB4
is able to induce a rapidly occurring
but transient
monolayers. The effect of LTB4 is specific, since the structural
which differs from LTB4 with regard to cis-trans configurations and has
no neutrophil activating effect (12), was ineffective in the here described system. In previous studies LTB4 has been reported either to lack or to possess an adherence promoting activity on endothelial
cells (4,13). As shown here, the transient nature of the LTB4 response
required careful evaluation of early time points to be detected. In contrast, responses to thrombin and PAF, and particularly to PMA, emerged slowly and were protracted. The maximal responses to equimolar concentrations of PAF and PM.4 were stronger than those to LTB4 (and thrombin). Some of these response characteristics have been reported
previously but no comparisons have
appeared.
Table
1.
PMN hypemdhesiveness
HUVEC treated with
% hyperadherence
to HVVEC
n
LTB4, loo nM
+10.3*1.3
17
SS,12SdiHETE, 100 nM
-1.7f2.0
3
Mean and SE values for hyperadherenm of PMNs (i.e. the increase of adherence above HBSStreated controls). HUVEC monolayers were treated with the stimuli for 5 mitt, washed, and PMNs were added. Hyperadherence was assessed5 min later. n=number of separate experiments, run in dupli- or triplicates. The negative value for SSJ2SdiHETE denotes that adherence values were lower than for HBSS stimulated controls.
850
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The rapid kinetics of the LTB4 adhesion response is similar to what we have observed LTB4 induced change of the membrane potential of HUVEC concentration
(6) as well as the cytosolic calcium
changes, aggregation and superoxide ion production
(7-9). This unique property of LTB4
for the
of neutrophils
and eosinophils
is probably expressed at a step of the stimulus response
coupling that is close to the surface receptor since the LTB4 induced generation of diacylglycerols and inositol phosphates exhibit similar kinetics (14,lS). It is not known whether HUVEC
express
surface receptors for LTBI ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (19X-05991,19P7fl95), The Swedish Association against Rheumatism, King Gustaf v’s SO- year Fund, the Funds of the Karolinska Institute, Sodersjukhuset, P&A Hedlund, S & E-C Hagberg, The Swedish Medical Society and Wrenade Liv Insurance Comp. The skilful technical assistance of Mrs. S. Myrin, P. Spingberg and Mr. C. Forsbom is gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Zimmerman, GA., Whatley, R.E., McIntyre, T.M. and Prescott, SM. (1987) Am. Rev. Resp. Dis. 136,204-207. Webster, R.0, Wysolmerski, R.B. and Lagunoff,D. (1986) Am. J. Pathol. 125369-378. Schleimer, R.P. and Rutledge B.K (1986) J. Immunol. 136,649-654. McIntyre, T.M., Zimmerman, GA and Prescott, SM. (1986) Proc. Natl. Acad. Sci. USA 83, 2204-2208. Garcia, J.G.N., Azghani, A, Callahan, KS. and Johnson, A.R. (1988) Thrombosis Res. 51, 83-96. Werner, R., Lindstrom, P. and Palmblad, J. (19SS) Biochem. Biophys. Res. Commun. 153, 805-810. Palmblad, J., Gyllenhammar, H., Lindgren, J.A. & Malmsten, C.L. (1984) J. Immunol. 132, 3041-3045. Palmblad, J., Gyllenhammar, H., Ringertz, B., Nilsson, E. and Cottell, B. (1988) Biochim. Biophys. Acta 970,92-102. Ringer@ B., Palmblad, J. and Lindgren, J.k (1985) J. Lab. Clin. Med. 106,132-140. Jaffe, EA., Nachman, R.L. Becker, C.G. and Miniek, R. (1973) J. Clin. Invest. 52,2745-2756. Lindstrom, P. and Palmblad, J. (1988) J. Clin. Lab. Immunol. 25,77-81. Palmblad, J, Uden, A-M., Lindgren, J.A., R&dmark, O., Hanson G. and Malmsten, C.L. (1982) FEBS Lett. 144,Sl-83. Hoover, R.L., Kamovsky, M.J., Austen, RF., Corey, E,J, and Lewis, R.A. (1984) Proc. Natl. Acad. Sci. USA 81,2191-2193. Riebman, J., Korchak, H.M., Vosshall, L.B., Haines, KA., Rich, AM. and Weissmann, G. (1988) J. Biol. Chem. 263,6322632X Truett, AP. III, Verghese, M.W., Dillon, S.B. and Snyderman, R. (1988) Proc. Natl. Acad. Sci. USA S5,1549-1553.
851
