- Email: [email protected]
Lymphocyte interacting adhesion molecules on brain microvascular cells
0161-5890/90$3.00+ 0.00 Pergamon Press plc
MolecularImmunolog)?, Vol. 27, No. 12, pp. 1355-1359,1990 Printed in Great Britain.
LYMPHOCYTE INTERACTING ADHESION MOLECULES ON BRAIN MICROVASCULAR CELLS MICHAEL N. HART,* ZSUZSANNAFABRY, MARI WALDSCHMIDTand MATYAS SANDOR Department of Pathology, Division of Neuropathology, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. (First received 21 February 1990; accepted 21 March 1990)
INTRODUCTION Endothelial cells (En) throughout the body relate to immunocompetent hematopoietic cells in very secial and specific ways. For example, endothelial cells express adhesion molecules such as ICAM- 1, ELAM, LFA-3 (Bierer and Burakoff, 1988; Pober, 1988) which bind lymphocytes and other immunocompetent cells to endothelium with their respective ligands, presumably as a prelude to exiting from the vascular compartment into various organs and tissues (Cotran, 1987). The expression of these adhesion molecules is regulated by certain cytokines, including IL-l, TNF, IFN-1/ and others (Pober, 1988). It is also increasingly suspected that vascular smooth muscle (SM) cells may play a role in determining which hematopoietic cells may exit from the vascular compartment under given conditions (Pardridge et al., 1989). MHC II expression has also been shown on En and SM cells (Hart et al., 1987) and suggested to be important under pathological conditions (Pollard et al., 1986; Jonasson et al., 1988). Further, En and vascular SM cells may behave differently in different areas of the body. For example, it is known that there are differences between large and small muscled En and SM, and it is increasingly suspected that En, at least from various organs, differ considerably from each other. In spite of the existence of immunological reactions in brain and their role in development of brain diseases, there is a very small body of data suggesting the immunological properties of brain En and SM cells. En cells from the brain display significantly different morphology from other En cells, having tight junctions between them and a paucity of micropinocytotic vesicles. It is this morphology which is associated with the bloodbrain barrier, a barrier for both cellular elements and macromolecules in the central nervous system (Fenstermacher and Rapoport, 1984). The purpose of this report is to summarize the studies which we have performed in our laboratory regarding the interactions between the elements of the brain microvessel and lymphoid systems. *Author to whom correspondence
should be addressed.
We observed several years ago that BALB/c splenic cells co-cultured with irradiated BALB/c SM cells for one week would become activated and proliferate (Hart et al., 1985). When lo6 of these activated cells were injected intravenously into a syngeneic host, a vasculitis resulted which was often destructive of SM elements in small and medium sized arterioles and venules. The experiments that ensued following this finding were designed to address two general sets of questions. The first set of questions related to events taking place in the affector aspect of the model. That is, what caused the lymphocytes to become activated when in contact with SM? Is SM an antigen presenting cell or an accessory bystander cell? Do En cells relate to lymphoid cells in the same manner that SM cells do? Is MHC expressed on these cells, and is the interaction between lymphoid cells and En and SM cells MHC-restricted? We also wanted to know which types of lymphoid cells were being activated by the SM cells. The second set of questions that we asked addresed the effector aspects of the model. For example, what type of cell is eliciting the damage in the vasculitis? How do these immunocompetent cells relate to the En on their way to damaging the SM? What types of adhesion molecules are elicited by En cells under these circumstances, and how are these adhesion molecules regulated? For all of our experiments we have utilized En and SM cells derived from the brain of BALB/c (H2d) and/or SJL/j (H2”)‘sources. These cells have been isolated and maintained by previously described methods (DeBault et al., 1981; Moore et al., 1984). Using the fluorescence activated cell sorter (FACS) we are able to utilize cultures of En or SM cells that are in the range of 96-98% pure using the lectin Griffonia Simplicifolia Agglutinin for identification of the En cells and an anti muscle-specific actin for identification of the SM cells (Sahagun et al., 1989). MHC-II cular SM
expression
and regulation
on brain microvas-
and En cells
The constitutive expression of Ia molecules on the surface of SM cells was published previously by us (Hart et al., 1987). Our results showed a significantly greater expression of Ia on SM cells (22-35%) in 1355
M. N. HAKTet al.
--___
I
P
d p I E L. d
20000
10000
m’
MK-D6
I
0 OVA
U\ml
0
recombinant
I
murine
2
3
Fig. 2. OVA antigen presentation by cultured brain SM cells. Irradiated spleen cells (1 x 105), brain SM or EN cells were incubated with 1 x 10’ OVA specific T cells (A2.2ElO) in the presence of 100 pg of whole or digested OVA (dOVA) as an antigen, IL2 production of T cells was checked after 2 days incubation using CTLL IL2-dependent cell line. [IH]thymidine incorporation of the CTLL cell line in the presence of culture supernatant is shown. The results show A mean CPM i. S.D. of triplicates in three experiments.
IFN-gamma
4
5
Days
Fig. I. Effect of murine recombinant IFN-y on the expression of MHC II molecules on brain vascular SM cells. SM cells were treated with IFN-y at the indicated concentrations for 72 hr (1A) or exposed to IFN-7 (125 U/ml) for the indicated time periods (1B). MHC II molecule expression was detected using saturated amounts of biotinylated monoclonal antibodies and fluorescence labeled Avidin. Cell surface staining was quantified using a FACSIV system, data were collected into a VAX computer, and analyzed by a DESK program. In all experiments, propidium iodide was added to identify and exclude dead cells. Fluorescent avidin alone stained less than 5% of the cells and was subtracted from the percentage of the positive cells.
contrast to En (S-10%). Here we report that recombinant murine IFN-y augments the expression of MHC II molecules on the SM cells with 100 U/ml being the optimal concentration {Fig. iA), and 3 days being the time of optimal response (Fig. 13).
unstimulated brain SM cells are able to present antigen to A2.2ElO cells. Whole OVA and digested OVA were presented by brain SM cells equally well. In contrast, unst~mulated brain En cells presented OVA and digested OVA at a much lower level than SM to these T cell hybridoma cells. Spleen cells were used as positive sontrols. In order to determine whether SM cell antigen presentation was limited to OVA, we tested them in another antigen presentation system, where KLH was used as antigen recognized by HDK-1, a KLH specific, H2d restricted T cell clone. As seen in Fig. 3, SM cells could present KLH antigen to the HDK-1 T cell clone, detected by the increased proliferation of the HDK-1 T cells. The antigen presenting capacity of En cells in the HDK1 KLH assay was again much lower compared to spleen or SM cells. The antigen presenting capacity of the SM cells was remarkably augmented by IFN-y whereas the antigen presenting capacity of En was only slightly increased by IFN-g (Fig. 4). This effect could be due to increased expression of Ia or IFN-y inducible co-stimulatory activity in SM or En cells. MHC class II dependency and restriction in this system was shown by successful blocking of antigen presentation with relatively low doses of an anti-fa
Functional role of MCH II molecules expressed on SM cells
Because it is generally accepted that expression of Ia molecules on the surface of a given cell is necessary but not sufficient for antigen presentation by that cell type, we further investigated the functional role of MHC II molecules on SM cells in antigen presentation. OVA and KLH were given to Sm and En as exogenous antigens, presented to OVA-specific H2d restricted A2.2El0, a T cell hybridoma, or to a KLH-specific H2d restricted T cell clone (HDK-1). A22ElO cells are known to produce IL-2 after stimulation with OVA presented in the context of H2d MHC (Glimcher and Shevach, 1982). IL-2 production from the supernatants was tested using an IL-2 dependent CTLL cell line. Figure 2 shows that
Fig. 3. KLH antigen presentation by cultured brain SM cells. Irradiated spleen ceils (1 x IO’). SM or En cells were incubated for 2 days with I x lo5 KLH-recognizing T cells (HDK) in the presence of 100pgjml KLH antigen. pH]thymidine incorporation of the T cells after 2 days culture was detected as described in Materialsand Methods. The results show A mean cpm & SD. of triplicates in three experiments.
Lymphocyte adhesion molecules on brain cells
P i
40000
1357
SM
30000
f 2 r
20000
= -d
10000
Rd
-E 01 0
100
200 IFN-gamma
300
400
500
U\ml
Fig. 4. Effect of IFN-11 on antigen-presentation by brain SM and En cells. Brain SM and En cells were pre-incubated with varying concentrations of IFN-), for 3 days prior to antigen presentation assay which was carried out as descried in Materials and Methods using KLH antigen and HDK-1
clone. Results show A mean f S.D. of antigen-specific T cell proliferation (n = 3). monoclonal antibody that recognizes both IAd and IEd (M5.114) and also complete blocking, but at higher doses, by anti-IAd antibody (MK-D6). AntiMHC II (34-I-29) had no effect on antigen presentation (Fig. 5). These results show that microvessel SM from the brain is an effective antigen presenting cell and can process antigen. Further, the antigen presenting capacity of SM is augmented by IFN-y and blocked by anti-Ia. ICAM-I
expression on bruin En
Although antigen-specific activation of the T cell is mediated by the TcR-CD3 complex, a number of other surface molecules participate in the immune response. These molecules may also play a role in lymphocyte migration, homing and recirculation. The next part of our paper will focus on the expression of intercellular adhesion molecule-l (ICAM-1)-a ligand for lymphocyte function associated antigen- 1 (LFA-1). ICAMis a widely distributed molecule, expressed on lymphoid and non-lymphoid cells including vascular endothelial cells, thymic epithehal
Fig. 5. Effect of anti-MHC II monoclonal antibody on the antigen presentation by brain SM cells. Irradiated brain SM cells (1 x 105) were incubated with 110 pg OVA antigen and I x lo5 A2.2ElO antigen specific T cells in the presence of anti I-A” (MKD6), anti I-Ad, I-Ed (M5.114), or anti H-2 KdDd (34-I-23) monoclonal antibodies. After 2 days, IL2 production of A2.2ElO cells was measured and compared to control cultures (antigen presentation by SM cells without anti-MHC monoclonal antibodies) (A mean f S.D., n = 3).
cells, mucosal epithelial cells, and dendritic cells. Its expression is uniformly low on peripheral leukocytes, but high on EBV-transformed B lymphoblasts and on mitogen-stimulated T lymphoblasts. Figures 6 and 7 show that ICAMmolecules are expressed on brain microvessel En cells, however there is very little, if any, expression on brain SM cells. A 3T3 fibroblast cell line was used as a known positive. Since ICAM- 1, LFA-I interaction is thought to be important in antigen presentation and other cellcell interactions (Boyd et al., 1988) it is very interesting that microvesse1 brain SM expresses ICAMto a much lesser degree and yet is a much more effective antigen presenting cell than the En which appears to express large amounts of ICAM-1. This finding raises the possibility that other ligands exist for LFA-1 molecules besides ICAM-1, which has also been suggested by others (Bierer and Burakoff, 1988).
Fc receptors in the blood-brain barrier system Besides the MHC II or adhesion molecules, other cell surface structures on En or SM cells can play a
ICAM I Fig. 6. ICAM-I expression on brain microvascular cells. ICAMmolecule expression was detected by YNl/1.7 monoclonal antibody, kindly provided by Dr Fumio Takei, and using FITC anti-rat immunoglobulin (Cappel) as a secondary reagent. Cell surface staining of 3T3 fibroblasts (Panel A), En cells (Panel B) or SM cells (Panel C) was quantified using a FACS-IV system. Data were collected into a VAX computer and analyzed by a DESK program. In all experiments, propidium iodide was added to identify and exclude dead cells. FITC anti-rat immunologlobulin alone stained less than 5% of the cells (First peaks in each panel).
M. N. HART et al
1358
ICAM I
Fig. 7. Increased ICAM-I expression on ILla, IFN-y and TNFa treated En measured by immunofluorescence flow cytometry. En cells (106) were incubated for 1, 2, 4 or 18 hr before harvesting and processing for flow cytometry with 5 U/ml recombinant murine IL-l (Panel A), 100 U/ml recombinant murine IFN-y (Panel B) and 100 U/ml recombinant murine TNFa (Panel C). Cell surface staining was detected by YN1/1.7 monoclonal antibody and FITC anti-rat immunoglobulin. FITC anti-rat immunoglobulin alone stained less than 5% of the cells (first peak in each panel). role in interactions culature
and
between
immune
elements
systems.
More
of the microvasrecent
exper-
in our laboratory have addressed the presence of Fc receptors (FcR) on brain cells. Using both aggregate IgG and a monoclonal antibody that recognizes FCy R II (2.402) it was shown that BALB/c En and SM both constitutively express FcR, but that the expression is greater on En (Fig. 8) It was also shown that although astrocytes do not express FcR
iments
in early passes, this expression is increased with time and with passage. Oligodendroglia did not ever express FcR (data not shown). Correlating well with these in vitro findings was the demonstration of FcR expression in En, Sm and astrocytes using the 2.462 antibody with a peroxidase labeling system in frozen brain slices (not shown). Evidence that the presence of FcR on brain En may have a functional or physiologic role comes from the observation that
_~ ‘e-f-+ J
I
10
00
‘J
‘:““.“I GSA- FITC
GSA-FITC
a-actin_FITC
a-octiwFITC
Fig. 8. Expression of FcyR on brain microvascular cells. FeyRI1 expression on brain En cells (upper panels) or on brain SM cells (lower panels) has been detected by two color cytofluorometry. The probes for Fey R detection have been Avidin-Texas red following the staining of the cells with biotinylated 2.462 monoclonal antibody (middle panels) or by biotinylated mouse aggregated IgG (right panels). Biotinylated En cells have been co-stained with En specific GSA-FITC lectin, while the SM cells have been labeled by FITC conjugated anti-a actin antibody. The left panels are unstained controls. The figure shows that both En and SM cells express Fey receptors.
Lymphocyte
adhesion
25pg
$
5pg
125 pg 25lJg.
t
5pg
diseases involving
the
by NIH Veteran’s
F
REFERENCES
Medium
I
2 pMol/ml
3
PGEE
Fig. 9. Production of PGE, by brain En cells activated through their FeyR. Brain En cells were treated with indicated amounts of aggregated mouse IgG or 2.4G2 monoclonal antibody for 4 hr. PGE, production was measured using HPLC. Results show increased PGE, production following treatment with IgG or 2.462. of the FcR on En by either 2.462 or aggregate IgG results in enhanced production of prostaglandin E, over baseline controls (Fig. 9). Very recently there are more and more data contradicting the concept that the nervous system is inaccessible to immune reactivity. Astrocytes were shown to express Ia molecules and present antigen (Fontana et al., 1984; Fierz et al., 1984). Here we show that brain microvessel SM cells in vitro can express Ia and present antigen. SM Ia expression and antigen presentation can also be influenced by IFN-y treatment. Brain En and SM cells differ in regard to their antigen presenting capability and ICAMadhesion molecule expression. The [CAM-l molecule expression on En cells can be upregulated by IFN-y, ILla and TNFa cytokines. FcyR can also be detected on the surface of brain microvascular cells, suggesting the possibility of regulating some En-SM functions by immunoglobulins or immunocomplexes. In fact, this has been shown by increased prostaglandin production by En cells after their activation with immunocomplexes. We are far from understanding completely the complex interactions between the elements of the microvascular and immune systems. All this data indicate that what was once thought to be, at best, passive interaction between lymphoid cells traversing through the vessels of the brain and the constituents of the blood vessel wall, is now being increasingly recognized as a controlled biological interaction that is carefully regulated. Further, the dogma that the brain is inaccessible to immune reactions needs to be replaced with a more dynamic model including all the interacting molecules and mediator produced by brain or immune cells. The aberration of this regulation might well prove to be an important mechanism in the induction of certain stimulation
1359
cells
Acknowledgements-This research was supported grants HL31944, HL14230 and NS24621 and Administration Research Funds.
F
1 JJg
2
on brain
inflammatory and autoimmune central nervous system.
125 119 B
molecules
Bierer B. E. and Burakoff S. J. (1988) T cell adhesion molecules. FASEB J. 2, 2584-2590. Boyd A. W., Wawryk S. O., Burns G. F. and Fecondo J. V. (1988) Intercellular adhesion molecule 1 (ICAM-1) has a central role in cell-cell contact-mediated immune mechanisms. Proc. Natn. Acad. Sri. U.S.A. 85, 3095-3099. Cotran R. S. (1987) New roles for the endothelium in inflammation and immunity. Amer. J. Pathol. 129, 407413. DeBault L. E., Henriquez E., Hart M. N. and Cancilla P. A. (1981) Cerebral microvessels and derived cells in tissue culture. II. Establishment, identification and preliminary characterization of an endothelial cell line. In Vitro 17, 480494. Fenstermacher J. D. and Rapoport S. I. (1984) Blood-brain barrier. Handbook of Physiology-The Cardiooascular System, Vol. IV, Microcirculation, (Edited by Ranken E. M. and Michel C. C.), Part 2, Chap. 21. Amer. Physiol. Sot., Bethesda, MD. Fierz W., Endler B., Reske K., Wekerle H. and Fontana A. (1985) Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation. J. Immunol. 134, 3785-3793. Fontana A., Fierz W. and Wekerle H. (1984) Astrocytes present myelin basic protein to encephalitogenic T cell lines. Nature 307, 273-276. Hart M. N., Waldschmidt M. M., Hanley-Hyde J. H., Moore S. A., Kemp J. D. and Schelper R. L. (1987) Brain microvascular smooth muscle expresses class II antigens. J. Immunol. 138, 296&2963. Hart M. N.. Tassel1 S. K., Sadewasser K. L., Schelper R. L. and Moore S. A. (1985) Autoimmune vasculitis resulting from in vitro immunization of lymphocytes to smooth muscle. Amer. J. Pathol. 119, 448455. Jonasson L., Helm J. and Hansson G. K. (1988) Smooth muscle cells express Ia antigens during arterial response to injury. Lab. Incest. 58, 310-315. Moore S. A., Strauch A. R.. Yoder E. T., Rubenstein P. A. and Hart M. N. (1984) Cerebral microvascular smooth muscle in tissue culture. In Vitro 20, 512-520. Pardridge W. M., Yang J., Buciak J. and Tourtelotte W. W. (1989) Human brain microvascular DR-antigen. J. Neurosci Res. 23, 337-341. Pober J. S. (1988) Cytokine-mediated activation of vascular endothelium. Amer. J. Pathol. 133, 426433. Pollard J. D., McComb P. A., Baverstock J.. Gatenby P. A. and McLeod J. G. (1986) Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy. J. Neuroimmunol. 13, 123-134. Sahagun G., Fabry Z., Moore S. A., Schelper R. L. and Hart M. N. (1989) Purification of murine endothelial cultures by flow cytometry using a fluorescent-labeled Griffonia Simplicifolia Agglutinin. Amer. J. Pathol. 134, 1227-1232.
MolecularImmunolog)?, Vol. 27, No. 12, pp. 1355-1359,1990 Printed in Great Britain.
LYMPHOCYTE INTERACTING ADHESION MOLECULES ON BRAIN MICROVASCULAR CELLS MICHAEL N. HART,* ZSUZSANNAFABRY, MARI WALDSCHMIDTand MATYAS SANDOR Department of Pathology, Division of Neuropathology, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. (First received 21 February 1990; accepted 21 March 1990)
INTRODUCTION Endothelial cells (En) throughout the body relate to immunocompetent hematopoietic cells in very secial and specific ways. For example, endothelial cells express adhesion molecules such as ICAM- 1, ELAM, LFA-3 (Bierer and Burakoff, 1988; Pober, 1988) which bind lymphocytes and other immunocompetent cells to endothelium with their respective ligands, presumably as a prelude to exiting from the vascular compartment into various organs and tissues (Cotran, 1987). The expression of these adhesion molecules is regulated by certain cytokines, including IL-l, TNF, IFN-1/ and others (Pober, 1988). It is also increasingly suspected that vascular smooth muscle (SM) cells may play a role in determining which hematopoietic cells may exit from the vascular compartment under given conditions (Pardridge et al., 1989). MHC II expression has also been shown on En and SM cells (Hart et al., 1987) and suggested to be important under pathological conditions (Pollard et al., 1986; Jonasson et al., 1988). Further, En and vascular SM cells may behave differently in different areas of the body. For example, it is known that there are differences between large and small muscled En and SM, and it is increasingly suspected that En, at least from various organs, differ considerably from each other. In spite of the existence of immunological reactions in brain and their role in development of brain diseases, there is a very small body of data suggesting the immunological properties of brain En and SM cells. En cells from the brain display significantly different morphology from other En cells, having tight junctions between them and a paucity of micropinocytotic vesicles. It is this morphology which is associated with the bloodbrain barrier, a barrier for both cellular elements and macromolecules in the central nervous system (Fenstermacher and Rapoport, 1984). The purpose of this report is to summarize the studies which we have performed in our laboratory regarding the interactions between the elements of the brain microvessel and lymphoid systems. *Author to whom correspondence
should be addressed.
We observed several years ago that BALB/c splenic cells co-cultured with irradiated BALB/c SM cells for one week would become activated and proliferate (Hart et al., 1985). When lo6 of these activated cells were injected intravenously into a syngeneic host, a vasculitis resulted which was often destructive of SM elements in small and medium sized arterioles and venules. The experiments that ensued following this finding were designed to address two general sets of questions. The first set of questions related to events taking place in the affector aspect of the model. That is, what caused the lymphocytes to become activated when in contact with SM? Is SM an antigen presenting cell or an accessory bystander cell? Do En cells relate to lymphoid cells in the same manner that SM cells do? Is MHC expressed on these cells, and is the interaction between lymphoid cells and En and SM cells MHC-restricted? We also wanted to know which types of lymphoid cells were being activated by the SM cells. The second set of questions that we asked addresed the effector aspects of the model. For example, what type of cell is eliciting the damage in the vasculitis? How do these immunocompetent cells relate to the En on their way to damaging the SM? What types of adhesion molecules are elicited by En cells under these circumstances, and how are these adhesion molecules regulated? For all of our experiments we have utilized En and SM cells derived from the brain of BALB/c (H2d) and/or SJL/j (H2”)‘sources. These cells have been isolated and maintained by previously described methods (DeBault et al., 1981; Moore et al., 1984). Using the fluorescence activated cell sorter (FACS) we are able to utilize cultures of En or SM cells that are in the range of 96-98% pure using the lectin Griffonia Simplicifolia Agglutinin for identification of the En cells and an anti muscle-specific actin for identification of the SM cells (Sahagun et al., 1989). MHC-II cular SM
expression
and regulation
on brain microvas-
and En cells
The constitutive expression of Ia molecules on the surface of SM cells was published previously by us (Hart et al., 1987). Our results showed a significantly greater expression of Ia on SM cells (22-35%) in 1355
M. N. HAKTet al.
--___
I
P
d p I E L. d
20000
10000
m’
MK-D6
I
0 OVA
U\ml
0
recombinant
I
murine
2
3
Fig. 2. OVA antigen presentation by cultured brain SM cells. Irradiated spleen cells (1 x 105), brain SM or EN cells were incubated with 1 x 10’ OVA specific T cells (A2.2ElO) in the presence of 100 pg of whole or digested OVA (dOVA) as an antigen, IL2 production of T cells was checked after 2 days incubation using CTLL IL2-dependent cell line. [IH]thymidine incorporation of the CTLL cell line in the presence of culture supernatant is shown. The results show A mean CPM i. S.D. of triplicates in three experiments.
IFN-gamma
4
5
Days
Fig. I. Effect of murine recombinant IFN-y on the expression of MHC II molecules on brain vascular SM cells. SM cells were treated with IFN-y at the indicated concentrations for 72 hr (1A) or exposed to IFN-7 (125 U/ml) for the indicated time periods (1B). MHC II molecule expression was detected using saturated amounts of biotinylated monoclonal antibodies and fluorescence labeled Avidin. Cell surface staining was quantified using a FACSIV system, data were collected into a VAX computer, and analyzed by a DESK program. In all experiments, propidium iodide was added to identify and exclude dead cells. Fluorescent avidin alone stained less than 5% of the cells and was subtracted from the percentage of the positive cells.
contrast to En (S-10%). Here we report that recombinant murine IFN-y augments the expression of MHC II molecules on the SM cells with 100 U/ml being the optimal concentration {Fig. iA), and 3 days being the time of optimal response (Fig. 13).
unstimulated brain SM cells are able to present antigen to A2.2ElO cells. Whole OVA and digested OVA were presented by brain SM cells equally well. In contrast, unst~mulated brain En cells presented OVA and digested OVA at a much lower level than SM to these T cell hybridoma cells. Spleen cells were used as positive sontrols. In order to determine whether SM cell antigen presentation was limited to OVA, we tested them in another antigen presentation system, where KLH was used as antigen recognized by HDK-1, a KLH specific, H2d restricted T cell clone. As seen in Fig. 3, SM cells could present KLH antigen to the HDK-1 T cell clone, detected by the increased proliferation of the HDK-1 T cells. The antigen presenting capacity of En cells in the HDK1 KLH assay was again much lower compared to spleen or SM cells. The antigen presenting capacity of the SM cells was remarkably augmented by IFN-y whereas the antigen presenting capacity of En was only slightly increased by IFN-g (Fig. 4). This effect could be due to increased expression of Ia or IFN-y inducible co-stimulatory activity in SM or En cells. MHC class II dependency and restriction in this system was shown by successful blocking of antigen presentation with relatively low doses of an anti-fa
Functional role of MCH II molecules expressed on SM cells
Because it is generally accepted that expression of Ia molecules on the surface of a given cell is necessary but not sufficient for antigen presentation by that cell type, we further investigated the functional role of MHC II molecules on SM cells in antigen presentation. OVA and KLH were given to Sm and En as exogenous antigens, presented to OVA-specific H2d restricted A2.2El0, a T cell hybridoma, or to a KLH-specific H2d restricted T cell clone (HDK-1). A22ElO cells are known to produce IL-2 after stimulation with OVA presented in the context of H2d MHC (Glimcher and Shevach, 1982). IL-2 production from the supernatants was tested using an IL-2 dependent CTLL cell line. Figure 2 shows that
Fig. 3. KLH antigen presentation by cultured brain SM cells. Irradiated spleen ceils (1 x IO’). SM or En cells were incubated for 2 days with I x lo5 KLH-recognizing T cells (HDK) in the presence of 100pgjml KLH antigen. pH]thymidine incorporation of the T cells after 2 days culture was detected as described in Materialsand Methods. The results show A mean cpm & SD. of triplicates in three experiments.
Lymphocyte adhesion molecules on brain cells
P i
40000
1357
SM
30000
f 2 r
20000
= -d
10000
Rd
-E 01 0
100
200 IFN-gamma
300
400
500
U\ml
Fig. 4. Effect of IFN-11 on antigen-presentation by brain SM and En cells. Brain SM and En cells were pre-incubated with varying concentrations of IFN-), for 3 days prior to antigen presentation assay which was carried out as descried in Materials and Methods using KLH antigen and HDK-1
clone. Results show A mean f S.D. of antigen-specific T cell proliferation (n = 3). monoclonal antibody that recognizes both IAd and IEd (M5.114) and also complete blocking, but at higher doses, by anti-IAd antibody (MK-D6). AntiMHC II (34-I-29) had no effect on antigen presentation (Fig. 5). These results show that microvessel SM from the brain is an effective antigen presenting cell and can process antigen. Further, the antigen presenting capacity of SM is augmented by IFN-y and blocked by anti-Ia. ICAM-I
expression on bruin En
Although antigen-specific activation of the T cell is mediated by the TcR-CD3 complex, a number of other surface molecules participate in the immune response. These molecules may also play a role in lymphocyte migration, homing and recirculation. The next part of our paper will focus on the expression of intercellular adhesion molecule-l (ICAM-1)-a ligand for lymphocyte function associated antigen- 1 (LFA-1). ICAMis a widely distributed molecule, expressed on lymphoid and non-lymphoid cells including vascular endothelial cells, thymic epithehal
Fig. 5. Effect of anti-MHC II monoclonal antibody on the antigen presentation by brain SM cells. Irradiated brain SM cells (1 x 105) were incubated with 110 pg OVA antigen and I x lo5 A2.2ElO antigen specific T cells in the presence of anti I-A” (MKD6), anti I-Ad, I-Ed (M5.114), or anti H-2 KdDd (34-I-23) monoclonal antibodies. After 2 days, IL2 production of A2.2ElO cells was measured and compared to control cultures (antigen presentation by SM cells without anti-MHC monoclonal antibodies) (A mean f S.D., n = 3).
cells, mucosal epithelial cells, and dendritic cells. Its expression is uniformly low on peripheral leukocytes, but high on EBV-transformed B lymphoblasts and on mitogen-stimulated T lymphoblasts. Figures 6 and 7 show that ICAMmolecules are expressed on brain microvessel En cells, however there is very little, if any, expression on brain SM cells. A 3T3 fibroblast cell line was used as a known positive. Since ICAM- 1, LFA-I interaction is thought to be important in antigen presentation and other cellcell interactions (Boyd et al., 1988) it is very interesting that microvesse1 brain SM expresses ICAMto a much lesser degree and yet is a much more effective antigen presenting cell than the En which appears to express large amounts of ICAM-1. This finding raises the possibility that other ligands exist for LFA-1 molecules besides ICAM-1, which has also been suggested by others (Bierer and Burakoff, 1988).
Fc receptors in the blood-brain barrier system Besides the MHC II or adhesion molecules, other cell surface structures on En or SM cells can play a
ICAM I Fig. 6. ICAM-I expression on brain microvascular cells. ICAMmolecule expression was detected by YNl/1.7 monoclonal antibody, kindly provided by Dr Fumio Takei, and using FITC anti-rat immunoglobulin (Cappel) as a secondary reagent. Cell surface staining of 3T3 fibroblasts (Panel A), En cells (Panel B) or SM cells (Panel C) was quantified using a FACS-IV system. Data were collected into a VAX computer and analyzed by a DESK program. In all experiments, propidium iodide was added to identify and exclude dead cells. FITC anti-rat immunologlobulin alone stained less than 5% of the cells (First peaks in each panel).
M. N. HART et al
1358
ICAM I
Fig. 7. Increased ICAM-I expression on ILla, IFN-y and TNFa treated En measured by immunofluorescence flow cytometry. En cells (106) were incubated for 1, 2, 4 or 18 hr before harvesting and processing for flow cytometry with 5 U/ml recombinant murine IL-l (Panel A), 100 U/ml recombinant murine IFN-y (Panel B) and 100 U/ml recombinant murine TNFa (Panel C). Cell surface staining was detected by YN1/1.7 monoclonal antibody and FITC anti-rat immunoglobulin. FITC anti-rat immunoglobulin alone stained less than 5% of the cells (first peak in each panel). role in interactions culature
and
between
immune
elements
systems.
More
of the microvasrecent
exper-
in our laboratory have addressed the presence of Fc receptors (FcR) on brain cells. Using both aggregate IgG and a monoclonal antibody that recognizes FCy R II (2.402) it was shown that BALB/c En and SM both constitutively express FcR, but that the expression is greater on En (Fig. 8) It was also shown that although astrocytes do not express FcR
iments
in early passes, this expression is increased with time and with passage. Oligodendroglia did not ever express FcR (data not shown). Correlating well with these in vitro findings was the demonstration of FcR expression in En, Sm and astrocytes using the 2.462 antibody with a peroxidase labeling system in frozen brain slices (not shown). Evidence that the presence of FcR on brain En may have a functional or physiologic role comes from the observation that
_~ ‘e-f-+ J
I
10
00
‘J
‘:““.“I GSA- FITC
GSA-FITC
a-actin_FITC
a-octiwFITC
Fig. 8. Expression of FcyR on brain microvascular cells. FeyRI1 expression on brain En cells (upper panels) or on brain SM cells (lower panels) has been detected by two color cytofluorometry. The probes for Fey R detection have been Avidin-Texas red following the staining of the cells with biotinylated 2.462 monoclonal antibody (middle panels) or by biotinylated mouse aggregated IgG (right panels). Biotinylated En cells have been co-stained with En specific GSA-FITC lectin, while the SM cells have been labeled by FITC conjugated anti-a actin antibody. The left panels are unstained controls. The figure shows that both En and SM cells express Fey receptors.
Lymphocyte
adhesion
25pg
$
5pg
125 pg 25lJg.
t
5pg
diseases involving
the
by NIH Veteran’s
F
REFERENCES
Medium
I
2 pMol/ml
3
PGEE
Fig. 9. Production of PGE, by brain En cells activated through their FeyR. Brain En cells were treated with indicated amounts of aggregated mouse IgG or 2.4G2 monoclonal antibody for 4 hr. PGE, production was measured using HPLC. Results show increased PGE, production following treatment with IgG or 2.462. of the FcR on En by either 2.462 or aggregate IgG results in enhanced production of prostaglandin E, over baseline controls (Fig. 9). Very recently there are more and more data contradicting the concept that the nervous system is inaccessible to immune reactivity. Astrocytes were shown to express Ia molecules and present antigen (Fontana et al., 1984; Fierz et al., 1984). Here we show that brain microvessel SM cells in vitro can express Ia and present antigen. SM Ia expression and antigen presentation can also be influenced by IFN-y treatment. Brain En and SM cells differ in regard to their antigen presenting capability and ICAMadhesion molecule expression. The [CAM-l molecule expression on En cells can be upregulated by IFN-y, ILla and TNFa cytokines. FcyR can also be detected on the surface of brain microvascular cells, suggesting the possibility of regulating some En-SM functions by immunoglobulins or immunocomplexes. In fact, this has been shown by increased prostaglandin production by En cells after their activation with immunocomplexes. We are far from understanding completely the complex interactions between the elements of the microvascular and immune systems. All this data indicate that what was once thought to be, at best, passive interaction between lymphoid cells traversing through the vessels of the brain and the constituents of the blood vessel wall, is now being increasingly recognized as a controlled biological interaction that is carefully regulated. Further, the dogma that the brain is inaccessible to immune reactions needs to be replaced with a more dynamic model including all the interacting molecules and mediator produced by brain or immune cells. The aberration of this regulation might well prove to be an important mechanism in the induction of certain stimulation
1359
cells
Acknowledgements-This research was supported grants HL31944, HL14230 and NS24621 and Administration Research Funds.
F
1 JJg
2
on brain
inflammatory and autoimmune central nervous system.
125 119 B
molecules
Bierer B. E. and Burakoff S. J. (1988) T cell adhesion molecules. FASEB J. 2, 2584-2590. Boyd A. W., Wawryk S. O., Burns G. F. and Fecondo J. V. (1988) Intercellular adhesion molecule 1 (ICAM-1) has a central role in cell-cell contact-mediated immune mechanisms. Proc. Natn. Acad. Sri. U.S.A. 85, 3095-3099. Cotran R. S. (1987) New roles for the endothelium in inflammation and immunity. Amer. J. Pathol. 129, 407413. DeBault L. E., Henriquez E., Hart M. N. and Cancilla P. A. (1981) Cerebral microvessels and derived cells in tissue culture. II. Establishment, identification and preliminary characterization of an endothelial cell line. In Vitro 17, 480494. Fenstermacher J. D. and Rapoport S. I. (1984) Blood-brain barrier. Handbook of Physiology-The Cardiooascular System, Vol. IV, Microcirculation, (Edited by Ranken E. M. and Michel C. C.), Part 2, Chap. 21. Amer. Physiol. Sot., Bethesda, MD. Fierz W., Endler B., Reske K., Wekerle H. and Fontana A. (1985) Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation. J. Immunol. 134, 3785-3793. Fontana A., Fierz W. and Wekerle H. (1984) Astrocytes present myelin basic protein to encephalitogenic T cell lines. Nature 307, 273-276. Hart M. N., Waldschmidt M. M., Hanley-Hyde J. H., Moore S. A., Kemp J. D. and Schelper R. L. (1987) Brain microvascular smooth muscle expresses class II antigens. J. Immunol. 138, 296&2963. Hart M. N.. Tassel1 S. K., Sadewasser K. L., Schelper R. L. and Moore S. A. (1985) Autoimmune vasculitis resulting from in vitro immunization of lymphocytes to smooth muscle. Amer. J. Pathol. 119, 448455. Jonasson L., Helm J. and Hansson G. K. (1988) Smooth muscle cells express Ia antigens during arterial response to injury. Lab. Incest. 58, 310-315. Moore S. A., Strauch A. R.. Yoder E. T., Rubenstein P. A. and Hart M. N. (1984) Cerebral microvascular smooth muscle in tissue culture. In Vitro 20, 512-520. Pardridge W. M., Yang J., Buciak J. and Tourtelotte W. W. (1989) Human brain microvascular DR-antigen. J. Neurosci Res. 23, 337-341. Pober J. S. (1988) Cytokine-mediated activation of vascular endothelium. Amer. J. Pathol. 133, 426433. Pollard J. D., McComb P. A., Baverstock J.. Gatenby P. A. and McLeod J. G. (1986) Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy. J. Neuroimmunol. 13, 123-134. Sahagun G., Fabry Z., Moore S. A., Schelper R. L. and Hart M. N. (1989) Purification of murine endothelial cultures by flow cytometry using a fluorescent-labeled Griffonia Simplicifolia Agglutinin. Amer. J. Pathol. 134, 1227-1232.