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From the National Public Health Institute and MediCity Research
Laboratory,*
the Departments of
Medicine,
Nuclear
Medicine,
and Pharmacology and Clinical
Pharmacology,§
the University of Turku, Turku,
Finland; MAP Medical Technologies Inc.,¶
Tikkakoski, Finland; the Cardiorespiratory Research
Unit,||
the Department of Surgery,**
and the Centre for Biotechnology,
Åbo Academy University and University of Turku, Turku, Finland; the
Department of Pathology,
Immunology
Division, Cambridge University, Cambridge, United Kingdom; the Orion
Corporation,§§
Orion Pharma, Turku,
Finland; and the Department of Nuclear
Medicine,¶¶
Turku University Central
Hospital, Turku, Finland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human vascular adhesion protein-1 (VAP-1), originally defined by 1B2 monoclonal antibody, is a 170-kd endothelial sialoglycoprotein.5 VAP-1 is inflammation inducible and mediates the early phases of interaction between lymphocytes and endothelium.6 The expression pattern of VAP-1 in normal and inflamed human tissues has been described7,8 and the role of VAP-1 in human leukocyte adhesion has been shown in vitro.5,9 However, practically nothing is known about the translocation of VAP-1 from the inside of the cells to the functional position on the cell surface as well as the significance of VAP-1 in leukocyte-endothelium interactions in vivo.
The anti-human-VAP-1 mAb 1B2 does not recognize VAP-1 of small laboratory animals such as mouse, rat, or rabbit. However, preliminary screening experiments revealed that 1B2 antibody does recognize porcine and canine blood vessels. That encouraged us to study whether the antigens recognized by 1B2 are the porcine and canine homologues of human VAP-1 and to develop inflammation models in these animals. The ultimate goal of this work was, for the first time, to gather novel data about the regulation of VAP-1 in vivo for further therapeutic use. Also as a direct clinical application of VAP-1 induction in diseased tissue we investigated whether VAP-1 can be used as a target for immunoscintigraphic imaging of inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse anti-human VAP-1 antibodies (1B25, IgM; 2D10,10 IgG1; TK8–148, IgG2a) and a mouse-human chimeric antibody, all against human VAP-1 were used to detect the porcine and canine forms of the antigen. The V-region domains of the chimeric anti-VAP-1 antibody were taken from TK 8–14 (Laukkanen et al, submitted). The control antibodies included 7C7, a mouse IgM monoclonal antibody that recognizes bursal epithelium of chicken; 3G6, a mouse IgG1 specific to chickens T cells;5 and 7E8, a mouse IgG1 against human TIE growth factor receptor.11 For the imaging experiments a nonbinding human chimeric antibody was engineered for use as a negative control. The antigen-binding site of the control antibody consists of domains from two different antibodies. The variable region of heavy chain is taken from an antibody against the hapten nitrophenylacetyl whereas the variable region of light chain is from an antilysozyme antibody. The constant region used in both the experimental VAP-1-specific and the control chimeric antibodies was a slightly modified form of human IgG2.12 In this constant region of the IgG2 residues A330 and P331 have been replaced by the residues S330 and S331 as found in human IgG4, and this has been shown to reduce the binding of the antibody to human Fc receptors and also to prevent human complement activation.12 For the canine experiments the antibodies were purified from serum-free culture supernatants by precipitation using ammonium sulfate. The mouse antibodies for pig experiments were produced in bioreactors and purified chromatographically as described.13 The chimeric antibodies were purified from the cell culture supernatants by using protein-A affinity chromatography. A peroxidase-conjugated goat anti-mouse Ig (DAKO, Glostrup, Denmark) and tetramethylrhodamine B isothiocyanate (TRITC)-conjugated goat anti-mouse IgM (Zymed, San Francisco, CA) antibody were used in the detection of mouse antibodies in immunohistochemistry. A mouse IgG1 antibody against porcine CD31 (Serotec Ltd., Oslo, Norway) and fluorescein isothiocyanate-conjugated F(ab')2 of sheep antibody against mouse IgG (Sigma Chemical Co., St. Louis, MO) were used to identify endothelial cells.
Radiolabeling of Antibodies
The chimeric anti-VAP-1 and control antibodies were labeled with I-123 and I-131, respectively, using the standard chloramine-T method. Briefly, an adequate amount of 123-I or 131-I in 100 to 150 µl of 0.18 mol/L phosphate buffer at pH 7.5 and 100 µg of antibody were mixed with 0.15 µg chloramine-T. After 5 minutes, the radiolabeled antibody was purified using PD-10 Sephadex G-25 size exclusion column (Pharmacia Biotech, Uppsala, Sweden) with 2% albumin/0.9% sodium chloride mobile phase. The purity of the radiolabeled immunoconjugate was determined by instant thin layer chromatography with 20% trichloro acetic acid as a solvent.
The biological activity of the labeled anti-VAP-1 antibody was studied from each labeling lot by an in vitro binding assay14 using VAP-1 and control-transfected Ax cells. The labeling method was adjusted to always leave the anti-VAP-1 antibody with a biological activity of >60%.
Immunohistochemistry
The tissue samples were immediately immersed in RPMI 1640 solution (Gibco BRL, Life Technologies Ltd., Paisley,UK), refrigerated, embedded in OCT compound (Tissue-Tec; Miles Inc., Elkhart, IN), frozen in liquid nitrogen, and, finally, serial sections were cut. The acetone-fixed sections were stained using immunohistochemical methods as described earlier.5 Briefly, sections were sequentially incubated for 40 minutes with 100 µl of primary mAb and appropriate peroxidase-conjugated secondary antibodies. The specimens from the animals that had received mouse antibodies intravenously were incubated with the peroxidase-conjugated anti-mouse Ig antibody. The immunoreaction was visualized using 3,3'-diaminobenzidine (Polysciences, Inc., Warrington, PA) as a chromogen. Finally, the sections were lightly counterstained with hematoxylin and mounted in DePeX (BDH, Poole, UK) for permanent records.
To allow better discrimination of the endothelium and other components of the vascular wall, a confocal microscope (Leica TSC 4D confocal system connected to Leica RXA microscope; Leica Microsystems Heidelberg GmbH, Heidelberg, Germany) was used. The specimens from the pigs that had received 1B2 or 7C7 intravenously were prepared in the following manner: 20-µm frozen sections were cut, air dried, fixed in -20°C acetone, and stored at -40°C. Sections were sequentially incubated with anti-CD31 and fluorescein isothiocyanate-labeled anti-mouse IgG or TRITC-labeled anti-mouse IgM secondary antibodies in phosphate-buffered saline containing 2% normal porcine serum. After the washes, the coverglasses were mounted with Fluoromount (Southern Biotechnology Associates, Inc., Birmingham, AL).
Immunoblotting
The samples from canine, porcine, and human peripheral lymph nodes, wall of gut (representing smooth muscle), and human tonsil tissues were lysed in 10 mmol/L Tris, pH 7.0, containing 0.15 mmol/L MgCl2, 5 mmol/L ethylenediaminetetraacetic acid, 2% Nonidet P-40, 1% Aprotinin (Sigma Chemical Co., St. Louis, MO), 1 mmol/L phenylmethylsulfonyl fluoride (Sigma), and 5 mmol/L NaN3 as described elsewhere.10 The centrifuged lysate was diluted in Laemmli sample buffer, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run in a standard way. The proteins were transferred from the gel to a Hybond nitrocellulose membrane (Amersham, Buckinghamshire, UK) with Hoefer Transphor Electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA). The membranes containing canine tissues were probed 1 hour with 1B2 or 7C7 antibodies (2 µg/ml). The bound primary antibodies were detected with the Vistra enhanced chemifluorescence Western blotting system (Amersham). FluorImager 595 (Molecular Dynamics, Sunnyvale, CA) was used to visualize the chemifluorescence. The membranes on which porcine VAP-1 was transferred were probed with TK8–14 and 3G6 antibodies, and the enhanced chemiluminescence (Amersham) system was used to visualize the human and porcine forms of VAP-1.
Inflammation Models
The skin inflammation was induced with 4 drops of 5% (w/v) dinitrochlorobenzene (1-chloro-2,4-dinitrobenzene, DNCB; Sigma) in acetone applied to 3 x 3-cm skin area. Control areas were painted with acetone only.
Aseptic arthritis was induced by intra-articular injection of 400 µg of phytohemagglutinin (Sigma) in 1 ml of RPMI 1640 8 hours before the administration of the radiolabeled antibody. Only RPMI 1640 was injected to the contralateral knee.
The design of the experimental inflammation studies and the respective
antibody treatment protocols are explained in detail in Figure 1
. All animal experiments were approved
by the Committee of Animal Care in Research of the University of Turku.
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From the pigs (receiving unlabeled antibodies) three blood samples
were collected in the beginning, at 60 minutes, and at the end of the
experiments to reveal the lowest antibody concentration during the
experiments. To determine the pharmacokinetics of the monoclonal
antibodies more precisely, three additional pigs received antibodies at
3 mg/kg (two 1B2 pigs, one 7C7 pig, experimental protocol III in Figure 1
) and blood samples were collected just before injection of antibody
at 2, 10, 20, 40, 60, 90, 120, 180, and 240 minutes after the
injection, and at the end of the experiment. The serum was separated
and the samples were stored at -40°C until analyzed. Concentrations
of the mouse antibodies were measured by time-resolved
immunofluorometric assay using microtitration wells coated with rabbit
anti-mouse immunoglobulin. The purified 1B2 and 7C7 mAb produced
earlier and the sera taken before antibody administrations were used as
standards. Europium-labeled sheep anti-mouse Ig (Boehringer-Mannheim,
Mannheim, Germany) and enhancement solution (Wallac, Turku, Finland)
were used for detection. The intensity of fluorescence was measured by
Fluorometer (Wallac). The results were converted to µg/ml using the
known standards. The pharmacokinetic modeling of the antibody
concentration data were done using two-compartment disposition
model.15
Imaging
During the induction of the joint inflammation and imaging the animals were kept under light anesthesia with ketamine hydrochloride (Ketalar, 50 mg/ml; Parke-Davis, Solna, Sweden) and diazepam (Stesolid Novum, 5 mg/ml; Dumex/Kabi Pharmacia, Sweden).
The single- and dual-isotope scintigraphies were performed with
Siemens Diacam (Siemens Gammasonics Inc., Des Plaines, IL) 
-camera.
Immediately after the intravenous administration of the labeled
chimeric antibodies dynamic imaging was started (3 hour imaging using
6 x 10 minutes and 6 x 20 minutes frames) and a 30-minute
static image was acquired 24 hours later. In the dual-isotope imaging
the energy windows were set as 159 keV (window width, 20%) for I-123
and 364 keV (15% window) for I-131. A third energy window for images
used in correction of the crosstalk and scatter artifact in
dual-isotope experiments was set as 210 keV (20% window). The
locations of the skin inflammations were marked in each series of the
images with a radiation source positioned outside the animals. At the
end of the last scan, the animals were humanely sacrificed with i.v.
administrations of pentobarbital (Mebunat, 60 mg/ml; Orion-Farmos,
Turku, Finland) and potassium chloride. To determine the kinetics of
the radiolabeled antibody accumulation, the regions of interest were
drawn on the areas of skin and joint inflammation and on corresponding
healthy control areas on the contralateral side. The 3-hour dynamic
image sets and the second day static images were visually analyzed for
maximal tracer uptake.
Tissue Distribution of Radiolabeled Antibodies
The tissues of the animals that had received radioactively labeled
antibodies were biopsied after the scintigraphies for detailed analysis
of radioactivity. Biopsies were obtained from the normal and inflamed
skin areas as well as from synovial membranes of inflamed and
uninflamed knees. The skin samples were obtained as a whole skin
thickness, whereas duplicate samples were taken from the most
superficial part of the skin containing only epidermis and part of the
dermis. The samples were weighed and the radioactivities of different
iodine isotopes were determined with a multichannel analyzator and
germanium crystal detector (Harshaw, Bicron, Washougal, WA) capable of
recognizing isotopes by their characteristic radiation spectra.
The results were converted to percentages of the injected dose per gram
of tissue and compared using Wilcoxon signed-rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the usefulness of dog and pig as animal models in which
to study the characteristics of VAP-1 in vivo we first
determined immunohistochemically the expression of VAP-1 in canine and
porcine tissues using 1B2 monoclonal antibody. The main results are
summarized in Table 1
. The expression of
VAP-1 in dog and pig closely resembles the published results for VAP-1
expression in humans.7
A baseline expression of VAP-1 was
seen in a subpopulation of small blood vessels in all of the tissues
studied. Moreover, smooth muscle cells of larger vessels are
VAP-1-positive although the endothelium of those blood vessels remains
VAP-1-negative. Also other smooth muscle cells as in the wall of the
gastrointestinal tract and in the myometrium are also VAP-1-positive.
1B2 also stains other cell types that may be considered related to
smooth muscle cells like pericytes of blood vessels and myoid cells of
testis. Heart and skeletal muscle cells, however, are negative for
VAP-1.
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To study the molecular nature of the canine and porcine VAP-1 more
precisely, the molecular weight of VAP-1 of dog and pig were studied by
immunoblotting and compared with that of humans. The molecular weights
of dog and pig VAP-1 are 10 to 20 kd smaller (Figure 2)
when compared with humans.
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The DNCB dosage that caused acute dermatitis categorized by
observation of erythema and edema but no bullae was used in all
experiments. After intravenous antibody administration, only the blood
vessels in the specimens from the inflamed skin of the 1B2-treated
animals bound the fluorescently- or peroxidase-labeled secondary
anti-mouse antibodies. The noninflamed skin and all other tissues of
the same animals remained completely negative. Also, no staining was
seen in the inflamed skin or in the other tissue specimens from the
animals treated with the control antibodies. These results indicate
that 1B2 antibody on the vessel wall was a result of specific binding
of the antibody in the areas of VAP-1 up-regulation and in noninflamed
areas VAP-1 remained completely intracellular and thus, nonaccessible
for the circulating antibody (Figure 3, a–f)
. Confocal microscopic studies showed that in some vessels the
anti-VAP-1 and anti-CD31 reactivities colocalized, whereas in some
other vessels the CD31 expression was more luminal than that of VAP-1
suggesting that in addition to endothelial VAP-1, the VAP-1-positive
pericytes/smooth muscle cells also contributed to the positive
immunoreaction seen in the blood vessels (Figure 3, g and h)
. The skin
inflammation model was also used to study the regulation of VAP-1
during inflammation (experimental protocol I, Figure 1
). The
translocation of VAP-1 to the cell surface was already seen in the skin
samples 60 minutes after the topical application of DNCB, the maximum
positivity was in the samples taken 8 hours after the induction, and
decreasing intensity of immunoreaction was observed at 24 and 48 hours
of inflammation.
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The pharmacokinetics of 1B2 and 7C7 antibodies were studied in
pigs. Complete sets of data allowing pharmacokinetic modeling were
obtained from three animals, two of which had received 1B2 and one 7C7
antibodies. The elimination curve of both antibodies was biphasic. A
pharmacokinetic curve after administration of 1B2 antibody is given in
Figure 4
. The half-lives of the initial
rapid distribution phase were 11, 26, and 8 minutes in the animals
which had received 1B2, 1B2, and 7C7, respectively. The corresponding
half-lives of the elimination phase were 240, 431, and 278 minutes. The
calculated apparent distribution volumes were 0.035 and 0.045 l/kg for
1B2 and 0.121 l/kg for the 7C7 antibody.
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Administration of both the chimeric anti-VAP-1 and control
antibodies resulted in enhanced tracer uptakes at the sites of
inflammation when compared with background activity. Based on the
immunostaining experiments described above (Figure 3)
, the accumulation
of the control antibody is considered to be an indicator of increased
blood flow as well as the endothelial leakage. The scintigraphies with
simultaneous administrations of chimeric anti-VAP-1 and control
antibodies and dual-label imaging protocol showed that specific
accumulation of the anti-VAP-1 antibody occurred at the areas of skin
and joint inflammation as well as in the liver. When the dynamic images
were analyzed, the accumulation of anti-VAP-1 antibody had a cumulative
trend at the inflamed areas whereas at the control areas the
radioactivity followed the radioactivity of blood (measured by drawing
regions of interest on the heart) in decreasing manner (data not
shown). As seen also in Figure 5
, in the
24-hour images the contrast between inflamed and noninflamed tissues is
better than in the first day images. Analysis of the radioactivity in
the skin and synovial samples, obtained immediately after 24-hour
imaging, showed that the accumulation of anti-VAP-1 antibody to sites
of inflammation was 10.38 ± 1.32 (mean ± SEM) times greater
than to comparable noninflamed control tissues. With the control
antibody the same ratio was 6.55 ± 1.06, respectively (Figure 6)
. There is a statistically significant
difference between the accumulation of anti-VAP-1 and control
antibodies at sites of inflammation (Wilcoxon signed-rank test,
P = 0.0019). In two additional animals that received
only chimeric anti-VAP-1 antibody, the accumulation to inflamed sites
was 9.8 ± 3.1 times greater than to noninflamed control areas.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was undertaken as part of an effort to find a suitable animal model for anti-adhesive drug development. Our working hypothesis was that the molecular characteristics and regulation of VAP-1 in man, dog, and pig would be comparable and these animal species could be used in such studies. The molecular weight of human VAP-1 is distinct in different organs and even in one organ under pathological conditions.25 The changes are believed to be because of alterations in carbohydrate structures of the molecules that are essential for the adhesive function of VAP-1.9 Indeed, in canine and porcine tissues the molecular weight close to that of human VAP-1 and the expression pattern of the antigen detected in immunohistochemistry in both species together suggest that the molecules recognized by mAb 1B2 in canine and porcine tissues really are the homologues of human VAP-1. VAP-1 has been found to be up-regulated in areas of inflammation when compared to the baseline expression.7,16,17 Importantly, in this present study, in vivo immunodetection experiments and immunohistochemical stainings showed that expression of VAP-1 on the luminal surface of endothelium is limited to areas of inflammation. These observations are consistent with the potential role of VAP-1 as a therapeutic target in inflammatory disorders and hold promise for relevance of our animal models for drug development purposes.
After translocation onto the cell surface, endothelial adhesion molecules may become shed or taken into various intracellular compartments.18-22 Our previous observations and current in vivo immunodetection experiments suggest that a significant proportion of up-regulated VAP-1 and anti-VAP-1 antibody complexes remain attached to the cell surface or become internalized.7,23 This observation forms the fundamental basis of using VAP-1 to target therapeutic compounds into inflamed tissues. In addition, we have shown here that VAP-1 can be used as a target in imaging inflammation. Radioactively labeled polyclonal (nonspecific) antibody can be used for that purpose because of the leakage of plasma proteins to extracellular space at sites of inflammation. Our results with the chimeric control antibody are consistent with that. However, when the ratios of the radioactivities in inflamed skin and synovium and healthy control specimens were studied, the amount of chimeric anti-VAP-1 antibody accumulation was approximately twofold higher when compared with the accumulation of the nonspecific antibody. The methodological strengths of our imaging study are that we were able to use a chimeric frame-matched control antibody in a dual-isotope protocol in which specific and nonspecific accumulation of the antibodies could be detected simultaneously. The dual-isotope scintigraphy is also a sophisticated way to overrule the biological variation between individual experiments. With these methods we were able to identify correctly the antigen-specific binding and nonspecific accumulation of the antibodies that otherwise inevitably complicates the interpretation of the results. Based on these results, anti-VAP-1 antibody is a more sensitive tool for imaging inflammation than a nonspecific antibody alone. Because the specific accumulation of anti-VAP-1 antibody may also happen in mild inflammation in which the mechanisms behind the nonspecific enhancement of the inflamed tissue may be less significant, we believe that using VAP-1 as a target in immunoscintigraphy deserves further studies.
Our present results show that circulating anti-VAP-1 antibody binds selectively to blood vessels in diseased tissue. However, besides its endothelial cell expression, VAP-1 is also present on the smooth muscle cells and pericytes of the vascular wall. The smooth muscle VAP-1 differs from endothelial VAP-1 in its carbohydrate modifications and it does not have adhesive function.10 Because the endothelium is permeable to the plasma proteins to a certain extent even in a noninflamed stage,24 the smooth muscle cells/pericytes of the vascular wall should bind anti-VAP-1 antibody if VAP-1 was on the cell surface. The fact that despite 6 hours of in vivo treatment with the 1B2 antibody the smooth muscle cells remain unstained in noninflamed tissues, suggests that the smooth muscle cells of the wall of blood vessels do not express VAP-1 on their surface in noninflamed conditions but, as in the endothelium, seem to relocate VAP-1 onto the cell surface in inflammation. This is especially beneficial for tissue targeting, because the blood vessels are capable of binding more diagnostic or therapeutic compounds to the inflamed tissue than in the case when only endothelial cells express the antigen.
In conclusion, our data show that VAP-1 in the pig and dog is an intracellular molecule in the noninflamed state and it is translocated onto the cell surface only after a proinflammatory stimulus. Therefore, VAP-1 well fills the requirements for a potentially effective target for anti-adhesive and diagnostic purposes. The results further indicate that the dog and pig can be used as experimental animals when biology of VAP-1 is studied.
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Acknowledgements |
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Footnotes |
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Supported by grants from Turku University Foundation, the Foundation of Aarne and Aili Turunen, the Finnish Academy, the Finnish Medical Society Duodecim, the Finnish Cultural Foundation, the Sigrid Juselius Foundation, Paulo Foundation, Instrumentarium Foundation, and the Finnish Foundation for Cardiovascular Research.
The current address of Tuomo Nikula is European Commission, Institute for Transuranium Elements, Karlsruhe, Germany; and the current address of Marja-Leena Laukkanen is VTT Biotechnology and Food Research, Espoo, Finland.
Accepted for publication April 23, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ki K, Vnttinen E, Saukko P, Alanen K, Kallajoki M, Voipio-Pulkki L-M, Salmi M: Vascular adhesion protein-1, ICAM-1 and P-selectin mediate leukocyte binding to ischemic heart in human. J Am Coll Cardiol 2000, 36:122-129This article has been cited by other articles:
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, Ratio of I-123-labeled anti-VAP-1 antibody per
gram inflamed tissue versus the same antibody per gram
comparable to noninflamed tissue of the same animal; 
, ratio of
I-131-labeled control antibody per gram inflamed tissue
versus the same antibody per gram comparable to noninflamed
tissue of the same animal.