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Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin 1 or tumor necrosis factor
Vo1,160, No. 3,1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1403-1408
May 15,1989
PRODUCTION OF MONOCYTE CHEMOTACTIC AND ACTIVATING FACTOR (MCAF) BY HUMAN DERMAL FIBROBLASTS IN RESPONSE TO INTERLEUKIN 1 OR TUMOR NECROSIS FACTOR
Christian G. Larsen, Claus O. C. Zachariae, Joost J. Oppenheim and Kouji Matsushima
Laboratory of Molecular Immunoregulation, Biological Response Modifiers program, Division of Cancer Treatment, National Cancer Institute, Frederick, MD 21701 Received April 6, 1989
Summary: Normal human dermal fibroblasts rapidly expressed (< 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL i) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type. ® 1989 AcademicPress, Inc.
Introduction:
The capacity of various types of cells to infiltrate and
accumulate at sites of inflammation suggests the existence of specific chemotactic cytokines.
The production of a soluble monocyte chemotactic
factor by a specific antigen- (purified protein derivative, PPD) or nonspecific mitogen-stimulated human peripheral blood leukocytes was first described by Snyderman et al (i).
They proposed that the production of
monocyte chemotactic factor by PPD-stimulated leukocytes is an in vitro correlate of delayed hypersensitivity. We have recently reported the purification of a novel basic heparinbinding monocyte chemotactic and activating factor (MCAF) from the conditioned media of a malignant human myelomonocytic cell line, THP-I (2) and molecularly cloned the cDNA for MCAF from HL-60 cell line cells (3).
MCAF is a potent
chemoattractant for monocytes in vitro and also augments the cytostatic activity of monocytes on several types of human tumor cell lines (2).
MCAF is
a member of the super-gene family to which the chemoattractant for neutrophils and lymphocytes, human interleukin 8 (IL 8) also belongs (4-7).
IL 8 can be
induced by stimulating human peripheral blood mononuclear cells, fibroblasts and keratinocytes with interleukin 1 (IL I) or tumor necrosis factor (TNF)
(5,8). 0006-291X/89
1403
$1.50
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
We have investigated whether non-malignant, non-circulating extravascular cells, e.g. fibroblasts and keratinocytes also produce MCAF in response to cytokines (i.e. IL i and TNF) and mitogens.
Materials and Methods: Cultures of human fibroblasts. Normal human dermal fibroblasts (32 SK, American Type Culture Collection, Rockville, MD) were cultured to subconfluency in tissue culture flasks (3150, Costar, Cambridge, MA) using 20 ml Dulbeccofs modified Eagle's medium supplemented with I0 % fetal bovine serum (Hyclone, Logan, UT), penicillin at I00 U/ml, streptomycin at i00 ug/ml and L-glutamine at 20 mM at 37°C and 5 % CO2 in air. Cells were stimulated by adding either recombinant IL i~ (rIL I~, 2x107 U/mg, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) or recombinant TNF~ (rTNF~, ixl07 U/mg, Dainippon Pharmaceutical Co. Ltd.) to the cultures at various concentrations and incubated for various time periods. Following incubation, the culture media were collected and prepared as described below. Total RNA was extracted from adherent fibroblasts as described previously (9). Cultures of normal human keratinocytes. Normal epidermal keratinocytes were obtained from Clonetics (San Diego, CA) and cultured in tissue culture flasks (3150,Costar) to subconfluency in 20 ml of the keratinocyte growth medium containing bovine pituitary extract, epidermal growth factor at I0 ng/ml, hydrocortisone at 0.5 ug/ml, insulin at 5 ug/ml, calcium at 0.15 mM and antibiotics. Keratinocytes were stimulated for 3 hrs with either rIL i~ or rTNF~. Following stimulation, both culture supernatants and cells were collected separately. Northern blotting analysis of mRNA for MCAF. Preparation of i0 ug total RNA from different cells were separated on electrophoreses, blotted onto nylon membranes and hybridized with 32p-labelled MCAF cDNA (0.65 kb Pst l-Pst 1 fragment) as described previously (4,9). The membranes were exposed to X ray films (X-OMAT-AR, Kodak, Rochester, NY) at -80°C for 16 hrs. Monocyte chemotactic assays for conditioned media. Monocyte chemotactic activity was measured using a modified multimicro-well Boyden chamber (i0) as described (2). Conditioned media from the cultures of dermal fibroblasts which were stimulated with either i00 U/ml IL i~ or TNF~ for 6 hrs were partially purified by a heparin Sepharose (Pharmacia, Uppsala, Sweden) as described (2). MCAF could be bound to a heparin Sepharose column which was equilibrated with 0.05 M Tris-HCl, pH 8.0 and eluted with 0.05 M Tris-HCl buffer, pH 8.0 with 0.75 M NaCI. Neither IL i~ or TNF~ was bound and recovered from a heparin Sepharose column under the same conditions.
Results:
We studied the capacity of IL i~ and TNF~ to induce the expression
of mRNA for MCAF in cultured normal human dermal fibroblasts. As shown in fig. la and fig. 2a both IL i~ at i00 U/ml and TNF~ at i00 U/ml induced very rapid expression of MCAF mRNA in dermal fibroblasts, i.e. within 30 min with maximal expression at 1-2 hrs.
Both cytokines proved to be potent MCAF inducers and
as little as 0.i U/ml induced a significant amount of MCAF mRNA.
IL I~ and
TNFa seems to be similar in potency with maximal MCAF induction at I00 U/ml at 2 hrs. (Fig ib and 2b). "In addition to investigating normal human dermal fibroblasts, we observed that a human fibrosarcoma cell line, 8387 (Ii) also expresses high levels of MCAF mRNA in response to IL Is or TNF~ (data not shown).
Since we had observed in our initial experiments that MCAF mRNA could
be highly induced by stimulating human peripheral blood mononuclear cells with
1404
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 160, No. 3, 1989
(a)
(b)
01/21
2
3
6 (hr)
0
0.1
1 10 100 1000 (Ulml)
\
~-28S
~--18S 0.7 Kb ----~
Time Course Experiment (100 U/mi)
Dose Response Experiment (3 hr)
Fisure i. MCAF mRNA induction in human dermal fibroblasts after the stimulation with recombinant IL Is. (a) Kinetics of response of fibroblasts to IL i~. (b) Dose response effect of IL is on the induction of MCAF mRNA at 3 hrs. Application of equal amounts of total RNA onto each lane was confirmed by running separate gels stained with ethidium bromide (data not shown). lipopolysaccharide positive
control
day old secondary various
(LPS), (fig. 2).
(SVK-14,
to IL is, TNF~,
a generous
We also measured cultures.
expression
an SV 40-transformed
London,
England)
Imperial
the release of monocyte
chemotactic
Cancer
activity from
Table i shows the levels of monocyte
stimulated,
and TNF~-stimulated index
fibroblasts.
chemotactic
activity was detected in culture media from fibroblasts
No significant monocyte
chemotactic
media from unstimulated
fibroblasts.
cells in the
cells present on
in medium alone per high power field.
6 hours with an optimal concentration
IL Is-
The results are given as
(CI)+ i SD, i.e. the number of migrating
samples divided by the number of migrating
incubated
cell
(data not shown).
chemotactic
filters
acid
failed to produce MCAF in response
activity in partially purified conditioned media from unstimulated,
experimental
to
of MCAF
human keratinocyte
gift from Dr. Taylor-Papadimitriou,
Ltd.,
IL is, or TNF~
fibroblast
from 7
interferon V, LPS, or phorbol myristic
Additionally,
Research Technology to LPS,
Likewise we tested if normal keratinocytes
We did not observe any significant
(data not shown). line
PBMC was used as
in vitro cultures expressed mRNA for MCAF in response
stimulants.
mRNA in response
total RNA from LPS-stimulated
Significant MCAF that were cultured for
of IL is (i00 U/ml)
or TNF~
(i00 U/ml).
activity was detected in the conditioned
1405
Vol, 160, No, 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (a)
(b)
o
L)
Q.
~3 o,. 0. .J
0 1/2 1 2 3 6 (hr)
0 0.1
1
10 100 1000
(U/ml)
•,
28S
-~----18S
0,7 Kb
~-
Time Course Experiment (100 Ulml)
Dose Response Experiment (3 hr)
Figure 2. MCAF mRNA induction in human dermal fibroblasts in response to human recombinant TNF~. (a) Rate of response of fibroblasts to I00 U/ml TNF~. (b) Dose response effect of TNF~ on the induction of MCAF mRNA at 3 hrs.
Discussion:
In the present report we have demonstrated that MCAF can be
produced by normal human dermal fibroblasts but not keratinocytes to two inflammatory cytokines,
IL in and TNF~.
in response
Recent preliminary experiments
showed that normal human endothelial cells also produce MCAF in vitro, when stimulated with IL In or TNF~ (C.G.L. et al., unpublished observation). Therefore,
the cell sources and regulation of MCAF production resembles that
of IL 8 production.
IL 8 has significant sequence homology with MCAF but has
distinct biological activity (5). cytokines,
The capacity of the proinflammatory
IL in and TNF~ to induce MCAF as well as IL 8 in various types of Table i
MONOCYTE CHEMOTACTIC ACTIVITY IN THE CONDITIONED MEDIA OF HUMAN DERMAL FIBROBLASTS STIMULATED WITH IL i or TNF Sources Medium control Unstimulated IL i stimulated TNF stimulated
CI Io00 1.41 3.87 4.74
SD 0.36 0.58 0.40 0.49
(p<0.001) (p<0.001)
Monocyte chemotactic activity was measured after partial purification by heparin Sepharose chromatography as described in the Materials and Methods. Normal human dermal fibroblasts were stimulated with either I00 U/ml IL is or TNF~ for 6 hrs. Cl=chemotactie Index, SD=standard deviation.
1406
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells suggests that some of the inflammatory reactions triggered by these cytokines could be mediated by these chemotactic polypeptides in vivo.
It
will be important to establish the production of these cytokines at different acute and chronic inflammatory sites (including sites of delayed hypersensitivity and granuloma).
When specific antibodies or other
antagonists to MCAF become available, it will be possible to directly examine the role of MCAF in these inflammatory reactions. It has been reported that various types of malignant tumor cells, such as ovarian carcinoma cells (ii), malignant melanoma cells (12), fibrosareoma cells (Ii), and glioblastoma cells (13) produce monocyte chemotactic factors in vitro, and that such tumors are often heavily infiltrated with monocytes in vivo which is associated with a better prognosis (12).
The capacity of tumors
to produce monocyte chemotactic factor inversely correlates with tumorigenicity in nude mice (12).
These data suggest that tumor infiltrating
monocytes may have an important role in regulating in vivo tumor cell growth. Therefore, MCAF may be clinically useful in increasing monocyte infiltration into tumor sites.
This idea is suggested by our recent observation that MCAF
also activates the cytostatic human monocyte activity against several types of human tumor cells in vitro (2).
The observation that intratumor injection of
TNF causes inflammatory leukocyte infiltration into tumors and is clinically more effective (14), may also partly be mediated by the production of IL 8 and MCAF.
Acknowledgments:
We are grateful to Drs. Kristian Thestrup-Pedersen,
Mukaida and Dan Longo for reviewing the manuscript.
Naofumi
Claus Zachariae was
supported by the Danish Medical Research Council Grant (No. 12-8261) and Mary Rosenkjaers Foundation.
References I. 2. 3.
4.
5.
6. 7.
Snyderman, R., Altman L.C., Hausman M.S., and Mergenhagen S.E. (1972) J. Immunol. 108, 857-860. Matsushima, K., Larsen, C.G., DuBois, G.C., and Oppenheim, J.J. (1989) J. Exp. Med. (in press). Furutani, Y., Nomura, H., Notake, M., Oyamada, Y., Fukui, T., Yamada, M., Larsen, C.G., Oppenheim, J.J., and Matsushima, K. (1989) Biochem. Biophys. Res. Commun. 159, 249-255. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E.A., Appella, E., Oppenheim, J.J., and Leonard, E.J. (1987) Proc. Natl. Acad. Sci. (USA) 84, 9233-9237. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H.-F., Leonard, E.J., and Oppenheim, J.J. (1988) J. Exp. Med. 167, 1883-1893. Larsen, C.G., Anderson, A.O., Appella, E., Oppenheim, J.J., and Matshushima, K. (1988) Science (in press). Zipfel, P.F., Balke, J., Irving, S.G., Kelly, K., and Siebenlist, U. (1989) J. Immunol. 142, 1582-1590.
1407
Vol. 160, No. 3, 1989
8. 9. i0. ii. 12.
13. 14.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Larsen, C.G., Anderson, A.O., Oppenheim, J.J., and Matsushima, K. (submitted for publication). Lew, W., Oppenheim, J.J., and Matsushima, K. (1988) J. Immunol. 140, 1895-1902. Falk, W., Goodwin, R.H., and Leonard, E.J. (1980) J. Immunol. Method. 33, 39-45. Ji-Ming, W., Cianciolo, G.J., Snyderman, R., and Mantovani, A. (1986) J. Immunol. 137, 2726-2732. Benomar, A., Ji-Ming, W., Taraboretti, G., Ghezzi, P., Bolotta, C., Cianciolo, G.J., Snyderman, R., Dore, J.-F., and Mantovani, A. (1987) J. Immunol. 138, 2372-2379. Kuratsu, J., Leonard, E.J., and Yoshimura, T. (1989) J. Natl. Cancer Inst. 81, 347-351. Taguchi, T. (1989) 2nd International Conference on Tumor Necrosis Factor and Related Cytokines. In Nappa, CA, USA. (Abst.) WA028.
1408
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1403-1408
May 15,1989
PRODUCTION OF MONOCYTE CHEMOTACTIC AND ACTIVATING FACTOR (MCAF) BY HUMAN DERMAL FIBROBLASTS IN RESPONSE TO INTERLEUKIN 1 OR TUMOR NECROSIS FACTOR
Christian G. Larsen, Claus O. C. Zachariae, Joost J. Oppenheim and Kouji Matsushima
Laboratory of Molecular Immunoregulation, Biological Response Modifiers program, Division of Cancer Treatment, National Cancer Institute, Frederick, MD 21701 Received April 6, 1989
Summary: Normal human dermal fibroblasts rapidly expressed (< 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL i) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type. ® 1989 AcademicPress, Inc.
Introduction:
The capacity of various types of cells to infiltrate and
accumulate at sites of inflammation suggests the existence of specific chemotactic cytokines.
The production of a soluble monocyte chemotactic
factor by a specific antigen- (purified protein derivative, PPD) or nonspecific mitogen-stimulated human peripheral blood leukocytes was first described by Snyderman et al (i).
They proposed that the production of
monocyte chemotactic factor by PPD-stimulated leukocytes is an in vitro correlate of delayed hypersensitivity. We have recently reported the purification of a novel basic heparinbinding monocyte chemotactic and activating factor (MCAF) from the conditioned media of a malignant human myelomonocytic cell line, THP-I (2) and molecularly cloned the cDNA for MCAF from HL-60 cell line cells (3).
MCAF is a potent
chemoattractant for monocytes in vitro and also augments the cytostatic activity of monocytes on several types of human tumor cell lines (2).
MCAF is
a member of the super-gene family to which the chemoattractant for neutrophils and lymphocytes, human interleukin 8 (IL 8) also belongs (4-7).
IL 8 can be
induced by stimulating human peripheral blood mononuclear cells, fibroblasts and keratinocytes with interleukin 1 (IL I) or tumor necrosis factor (TNF)
(5,8). 0006-291X/89
1403
$1.50
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
We have investigated whether non-malignant, non-circulating extravascular cells, e.g. fibroblasts and keratinocytes also produce MCAF in response to cytokines (i.e. IL i and TNF) and mitogens.
Materials and Methods: Cultures of human fibroblasts. Normal human dermal fibroblasts (32 SK, American Type Culture Collection, Rockville, MD) were cultured to subconfluency in tissue culture flasks (3150, Costar, Cambridge, MA) using 20 ml Dulbeccofs modified Eagle's medium supplemented with I0 % fetal bovine serum (Hyclone, Logan, UT), penicillin at I00 U/ml, streptomycin at i00 ug/ml and L-glutamine at 20 mM at 37°C and 5 % CO2 in air. Cells were stimulated by adding either recombinant IL i~ (rIL I~, 2x107 U/mg, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) or recombinant TNF~ (rTNF~, ixl07 U/mg, Dainippon Pharmaceutical Co. Ltd.) to the cultures at various concentrations and incubated for various time periods. Following incubation, the culture media were collected and prepared as described below. Total RNA was extracted from adherent fibroblasts as described previously (9). Cultures of normal human keratinocytes. Normal epidermal keratinocytes were obtained from Clonetics (San Diego, CA) and cultured in tissue culture flasks (3150,Costar) to subconfluency in 20 ml of the keratinocyte growth medium containing bovine pituitary extract, epidermal growth factor at I0 ng/ml, hydrocortisone at 0.5 ug/ml, insulin at 5 ug/ml, calcium at 0.15 mM and antibiotics. Keratinocytes were stimulated for 3 hrs with either rIL i~ or rTNF~. Following stimulation, both culture supernatants and cells were collected separately. Northern blotting analysis of mRNA for MCAF. Preparation of i0 ug total RNA from different cells were separated on electrophoreses, blotted onto nylon membranes and hybridized with 32p-labelled MCAF cDNA (0.65 kb Pst l-Pst 1 fragment) as described previously (4,9). The membranes were exposed to X ray films (X-OMAT-AR, Kodak, Rochester, NY) at -80°C for 16 hrs. Monocyte chemotactic assays for conditioned media. Monocyte chemotactic activity was measured using a modified multimicro-well Boyden chamber (i0) as described (2). Conditioned media from the cultures of dermal fibroblasts which were stimulated with either i00 U/ml IL i~ or TNF~ for 6 hrs were partially purified by a heparin Sepharose (Pharmacia, Uppsala, Sweden) as described (2). MCAF could be bound to a heparin Sepharose column which was equilibrated with 0.05 M Tris-HCl, pH 8.0 and eluted with 0.05 M Tris-HCl buffer, pH 8.0 with 0.75 M NaCI. Neither IL i~ or TNF~ was bound and recovered from a heparin Sepharose column under the same conditions.
Results:
We studied the capacity of IL i~ and TNF~ to induce the expression
of mRNA for MCAF in cultured normal human dermal fibroblasts. As shown in fig. la and fig. 2a both IL i~ at i00 U/ml and TNF~ at i00 U/ml induced very rapid expression of MCAF mRNA in dermal fibroblasts, i.e. within 30 min with maximal expression at 1-2 hrs.
Both cytokines proved to be potent MCAF inducers and
as little as 0.i U/ml induced a significant amount of MCAF mRNA.
IL I~ and
TNFa seems to be similar in potency with maximal MCAF induction at I00 U/ml at 2 hrs. (Fig ib and 2b). "In addition to investigating normal human dermal fibroblasts, we observed that a human fibrosarcoma cell line, 8387 (Ii) also expresses high levels of MCAF mRNA in response to IL Is or TNF~ (data not shown).
Since we had observed in our initial experiments that MCAF mRNA could
be highly induced by stimulating human peripheral blood mononuclear cells with
1404
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 160, No. 3, 1989
(a)
(b)
01/21
2
3
6 (hr)
0
0.1
1 10 100 1000 (Ulml)
\
~-28S
~--18S 0.7 Kb ----~
Time Course Experiment (100 U/mi)
Dose Response Experiment (3 hr)
Fisure i. MCAF mRNA induction in human dermal fibroblasts after the stimulation with recombinant IL Is. (a) Kinetics of response of fibroblasts to IL i~. (b) Dose response effect of IL is on the induction of MCAF mRNA at 3 hrs. Application of equal amounts of total RNA onto each lane was confirmed by running separate gels stained with ethidium bromide (data not shown). lipopolysaccharide positive
control
day old secondary various
(LPS), (fig. 2).
(SVK-14,
to IL is, TNF~,
a generous
We also measured cultures.
expression
an SV 40-transformed
London,
England)
Imperial
the release of monocyte
chemotactic
Cancer
activity from
Table i shows the levels of monocyte
stimulated,
and TNF~-stimulated index
fibroblasts.
chemotactic
activity was detected in culture media from fibroblasts
No significant monocyte
chemotactic
media from unstimulated
fibroblasts.
cells in the
cells present on
in medium alone per high power field.
6 hours with an optimal concentration
IL Is-
The results are given as
(CI)+ i SD, i.e. the number of migrating
samples divided by the number of migrating
incubated
cell
(data not shown).
chemotactic
filters
acid
failed to produce MCAF in response
activity in partially purified conditioned media from unstimulated,
experimental
to
of MCAF
human keratinocyte
gift from Dr. Taylor-Papadimitriou,
Ltd.,
IL is, or TNF~
fibroblast
from 7
interferon V, LPS, or phorbol myristic
Additionally,
Research Technology to LPS,
Likewise we tested if normal keratinocytes
We did not observe any significant
(data not shown). line
PBMC was used as
in vitro cultures expressed mRNA for MCAF in response
stimulants.
mRNA in response
total RNA from LPS-stimulated
Significant MCAF that were cultured for
of IL is (i00 U/ml)
or TNF~
(i00 U/ml).
activity was detected in the conditioned
1405
Vol, 160, No, 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (a)
(b)
o
L)
Q.
~3 o,. 0. .J
0 1/2 1 2 3 6 (hr)
0 0.1
1
10 100 1000
(U/ml)
•,
28S
-~----18S
0,7 Kb
~-
Time Course Experiment (100 Ulml)
Dose Response Experiment (3 hr)
Figure 2. MCAF mRNA induction in human dermal fibroblasts in response to human recombinant TNF~. (a) Rate of response of fibroblasts to I00 U/ml TNF~. (b) Dose response effect of TNF~ on the induction of MCAF mRNA at 3 hrs.
Discussion:
In the present report we have demonstrated that MCAF can be
produced by normal human dermal fibroblasts but not keratinocytes to two inflammatory cytokines,
IL in and TNF~.
in response
Recent preliminary experiments
showed that normal human endothelial cells also produce MCAF in vitro, when stimulated with IL In or TNF~ (C.G.L. et al., unpublished observation). Therefore,
the cell sources and regulation of MCAF production resembles that
of IL 8 production.
IL 8 has significant sequence homology with MCAF but has
distinct biological activity (5). cytokines,
The capacity of the proinflammatory
IL in and TNF~ to induce MCAF as well as IL 8 in various types of Table i
MONOCYTE CHEMOTACTIC ACTIVITY IN THE CONDITIONED MEDIA OF HUMAN DERMAL FIBROBLASTS STIMULATED WITH IL i or TNF Sources Medium control Unstimulated IL i stimulated TNF stimulated
CI Io00 1.41 3.87 4.74
SD 0.36 0.58 0.40 0.49
(p<0.001) (p<0.001)
Monocyte chemotactic activity was measured after partial purification by heparin Sepharose chromatography as described in the Materials and Methods. Normal human dermal fibroblasts were stimulated with either I00 U/ml IL is or TNF~ for 6 hrs. Cl=chemotactie Index, SD=standard deviation.
1406
Vol. 160, No. 3, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells suggests that some of the inflammatory reactions triggered by these cytokines could be mediated by these chemotactic polypeptides in vivo.
It
will be important to establish the production of these cytokines at different acute and chronic inflammatory sites (including sites of delayed hypersensitivity and granuloma).
When specific antibodies or other
antagonists to MCAF become available, it will be possible to directly examine the role of MCAF in these inflammatory reactions. It has been reported that various types of malignant tumor cells, such as ovarian carcinoma cells (ii), malignant melanoma cells (12), fibrosareoma cells (Ii), and glioblastoma cells (13) produce monocyte chemotactic factors in vitro, and that such tumors are often heavily infiltrated with monocytes in vivo which is associated with a better prognosis (12).
The capacity of tumors
to produce monocyte chemotactic factor inversely correlates with tumorigenicity in nude mice (12).
These data suggest that tumor infiltrating
monocytes may have an important role in regulating in vivo tumor cell growth. Therefore, MCAF may be clinically useful in increasing monocyte infiltration into tumor sites.
This idea is suggested by our recent observation that MCAF
also activates the cytostatic human monocyte activity against several types of human tumor cells in vitro (2).
The observation that intratumor injection of
TNF causes inflammatory leukocyte infiltration into tumors and is clinically more effective (14), may also partly be mediated by the production of IL 8 and MCAF.
Acknowledgments:
We are grateful to Drs. Kristian Thestrup-Pedersen,
Mukaida and Dan Longo for reviewing the manuscript.
Naofumi
Claus Zachariae was
supported by the Danish Medical Research Council Grant (No. 12-8261) and Mary Rosenkjaers Foundation.
References I. 2. 3.
4.
5.
6. 7.
Snyderman, R., Altman L.C., Hausman M.S., and Mergenhagen S.E. (1972) J. Immunol. 108, 857-860. Matsushima, K., Larsen, C.G., DuBois, G.C., and Oppenheim, J.J. (1989) J. Exp. Med. (in press). Furutani, Y., Nomura, H., Notake, M., Oyamada, Y., Fukui, T., Yamada, M., Larsen, C.G., Oppenheim, J.J., and Matsushima, K. (1989) Biochem. Biophys. Res. Commun. 159, 249-255. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E.A., Appella, E., Oppenheim, J.J., and Leonard, E.J. (1987) Proc. Natl. Acad. Sci. (USA) 84, 9233-9237. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H.-F., Leonard, E.J., and Oppenheim, J.J. (1988) J. Exp. Med. 167, 1883-1893. Larsen, C.G., Anderson, A.O., Appella, E., Oppenheim, J.J., and Matshushima, K. (1988) Science (in press). Zipfel, P.F., Balke, J., Irving, S.G., Kelly, K., and Siebenlist, U. (1989) J. Immunol. 142, 1582-1590.
1407
Vol. 160, No. 3, 1989
8. 9. i0. ii. 12.
13. 14.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Larsen, C.G., Anderson, A.O., Oppenheim, J.J., and Matsushima, K. (submitted for publication). Lew, W., Oppenheim, J.J., and Matsushima, K. (1988) J. Immunol. 140, 1895-1902. Falk, W., Goodwin, R.H., and Leonard, E.J. (1980) J. Immunol. Method. 33, 39-45. Ji-Ming, W., Cianciolo, G.J., Snyderman, R., and Mantovani, A. (1986) J. Immunol. 137, 2726-2732. Benomar, A., Ji-Ming, W., Taraboretti, G., Ghezzi, P., Bolotta, C., Cianciolo, G.J., Snyderman, R., Dore, J.-F., and Mantovani, A. (1987) J. Immunol. 138, 2372-2379. Kuratsu, J., Leonard, E.J., and Yoshimura, T. (1989) J. Natl. Cancer Inst. 81, 347-351. Taguchi, T. (1989) 2nd International Conference on Tumor Necrosis Factor and Related Cytokines. In Nappa, CA, USA. (Abst.) WA028.
1408