|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on May 24, 2002; DOI 10.1182/blood-2002-04-1017.
PHAGOCYTES
From the Rational Drug Design Program, Biomedicum
Helsinki; the Department of Bacteriology and Immunology and the
Department of Pathology, Haartman Institute, University of Helsinki;
Helsinki University Central Hospital (HUCS) Laboratory Diagnostics; and
the Department of Ophthalmology, Helsinki University Central Hospital,
Helsinki, Finland.
Hydrocortisone reduces the number of inflammatory leukocytes within
tissues, but thus far the site of action on the multistep adhesion
cascade leading to leukocyte extravasation has not been identified. We
have recently developed a noninvasive in vivo reflected-light confocal
microscopy technique to study this at sites of inflammation in human
patients. In the present study, we evaluated the effect of preoperative
intravenous hydrocortisone treatment on leukocyte trafficking after
conjunctival inflammation induced by cataract surgery in human subjects
in vivo. The surgery generated leukocyte rolling along the endothelial
lining of conjunctival vessels. While preoperative hydrocortisone did
not reduce the number of rolling cells, it significantly raised the
velocity of individual rolling leukocytes and concomitantly reduced
leukocyte emigration into conjunctival tissue. Immunohistology of
conjunctival biopsies excised from the individuals studied provided
circumstantial evidence that endothelial P-selectin might play a role
in the surgery-induced up-regulation of the leukocyte rolling.
Furthermore, hydrocortisone reduced surgery-induced P-selectin
induction, suggesting a role for this selectin in the regulation of
local leukocyte traffic into sites of inflammation in human
conjunctiva. Taken together, these results suggest that control of the
rolling velocity might be an effective way to adjust leukocyte traffic
in vivo in human subjects.
(Blood. 2002;100:2203-2207) Hydrocortisone has been used as an
anti-inflammatory drug for several decades,1,2 and we
asked whether its efficacy may be due to its modifying the multistep
leukocyte adhesion cascade, resulting in the generation of local
inflammatory lesions. If hydrocortisome is targeted toward leukocyte
trafficking, its anti-inflammatory effects can, at least theoretically,
result from changes in the number of rolling leukocytes or in
their rolling behavior or in the actual extravasation phase through the
interendothelial junctions.3-6
We have recently introduced a novel noninvasive application of
confocal reflected-light microscopy enabling direct and repeatable analysis and quantification of conjunctival inflammations in human patients.7 The human conjunctiva can be used in these
analyses, as it is semitransparent, and the tissue is normally devoid
of leukocytes, which greatly facilitates the identification of newly emigrated leukocytes during the inflammatory reaction. Standard phacoemulsification cataract surgery was used to induce a rapid and
very reproducible model of local conjunctival inflammatory reaction.
The hallmarks of this surgery-induced inflammation are (1) the rapid
increase in number of rolling leukocytes, which are almost exclusively
granulocytes, and (2) the velocity of these rolling leukocytes, which
is very slow, which is a prerequisite for (3) the extravasation of
leukocytes from the blood circulation into the conjunctival
tissue.7
In the present study, we evaluated the effect of preoperative
intravenous hydrocortisone treatment on leukocyte trafficking after
conjunctival inflammation induced by cataract surgery in human subjects
in vivo. The surgery generated leukocyte rolling along the endothelial
lining of conjunctival vessels. Whereas preoperative hydrocortisone did
not reduce the number of rolling cells, it significantly elevated the
velocity of individual rolling leukocytes and concomitantly reduced
leukocyte emigration into conjunctival tissue. Immunohistology of
conjunctival biopsies, excised from the individuals studied, suggested
that P- and L-selectin, but not E-selectin, might play a role in the
surgery-induced up-regulation of the leukocyte rolling. Furthermore,
hydrocortisone diminished surgery-induced P-selectin induction,
suggesting a role for this selectin on the regulation of local
leukocyte traffic into sites of inflammation in human conjunctiva.
Patients
The cataract operation was performed according to the standard
phacoemulsification technique under subtenon anesthesia. Half the
patients (4 of 8) received a single intravenous hydrocortisone injection (250 mg) 15 minutes before the operation. The surgery began
with a temporal conjunctival incision and cauterization of bleeding
episcleral vessels. The wounds were self-sealing without stitches, and
the conjunctiva was apposed to the wound by cautery. All patients
received 3 mg/mL topical ofloxacin (Exocin; Allergan, Irvine,
CA) as 1 to 2 drops × 4 for 3 preoperative days, and 1 mg/mL
dexametasone and 2 mg/mL chloramphenicol (Oftan Dexachlora; Santen,
Tampere, Finland) as 1 to 2 drops × 4 for 3 postoperative weeks.
In vivo confocal microscopy
Leukocyte rolling and histological image parameters Vessel diameters and the number of rolling cells were measured from all vessels. Mean centerline flow velocity was counted for 3 to 5 freely moving bright cells by measuring average movement in 4 subsequent frames. Vessels with flow above 500 µm/s were included in the analysis. The number of rolling cells was counted for each vessel with continuous flow and a sharp image from the cells passing an imaginary horizontal line in the vessel, which was fixed in one of the local landmarks in the vessel area to eliminate the effect of small movements. The number of extravasated leukocytes in the focal plane of the conjunctival stroma was counted from an area adjacent to the vessels where rolling had been analyzed.Immunohistochemistry The glycan epitopes on L-selectin ligands were identified by the monoclonal antibodies (mAbs) on formalin-fixed and paraffin-embedded conjunctival specimens. The mAb 2F3 (5 µg/mL)10 (Pharmingen, San Diego, CA) is anti-sialyl Lewis x (anti-sLex) mAb, and it requires presence of 2,3 sialylation and 1,3 fucosylation of the lactosamine. The
mAb MECA-79 (1:100 culture supernatant, from S. Jalkanen) requires
6-sulfation of the core 1 O-glycan decoration of L-selectin
ligands.11 We also used 10 µg/mL anti-vascular cellular
adhesion molecule-1 (anti-VCAM-1) mAb (1.4C3; Novocastra Laboratories,
Newcastle, United Kingdom). A polyclonal Ab was used against P-selectin
(10 µg/mL) (Pharmingen); E-selectin (5 µg/mL) (HP9017; HyCult,
Uden, The Netherlands); and intercellular adhesion molecule-1 (ICAM-1)
(5 µg/mL) (HP9018; HyCult). Isotype-matched mouse and rat
immunoglobulin G (IgG), IgM, and rabbit polyclonal Ab served as
negative control reagents with the same concentration on the same slide
(parallel section) as the specific Ab. Immunohistochemistry was
performed according to the relevant ABC Elite Kit protocols (Vector
Laboratories, Burlingame CA), with the use of pretreatment in
citrate buffer, pH 5, 2 × 5 minutes (mAbs 2F3 and MECA-79) or
at pH 3 (P- and E-selectin and ICAM-1), and incubation of
primary Ab overnight at 4°C (except E-selectin and
ICAM-1, which were incubated for 1 hour at room temperature
[RT]). A separate protocol for the anti-VCAM-1 mAb was used
according to the manufacturer's instructions (incubation of
primary mAb overnight at 4°C).
The reactivity of mAbs was evaluated by J.K., who had no knowledge of the pathological diagnosis of the specimens. The number of all vessels (CD34+ mAb) from a biopsy was calculated with the use of × 400 magnification to get the total number of vessels per section. The staining was scored either positive or negative for each vessel. To eliminate inherent variations between individuals in the number of vessels analyzed, the number of specific Ab-reactive vessels was divided by the number of CD34+ vessels from the same specimen (× 100%), yielding the percentage of specific Ab-reactive vessels in one patient. The mean ± SD was calculated from these normalized percentage values. E-selectin was not expressed in specimens taken either before or during inflammation, although we were able to induce a strong and specific staining in an inflamed skin biopsy specimen treated with 100 ng/mL lipopolysaccharide (LPS) and incubated in Dulbecco modified Eagle medium (DMEM) for 4 hours in 37°C in 5% CO2 atmosphere. Statistics The Mann-Whitney U test was used to compare the different groups (hydrocortisone treated versus untreated) against each parameter. The Wilcoxon signed rank test was used to compare the same patient group, for example, cortisone treated, at different time points. P < .05 was considered significant.
The cataract surgery in the 8 otherwise healthy individuals
induced a strong local inflammatory response in every patient, as
reported previously.7 To study the effects of
hydrocortisone administration on leukocyte trafficking, 4 of 8 patients
received a single preoperative intravenous hydrocortisone
injection. The analysis was performed so that the number of
vessels, the vessel diameter, analysis time, and hemodynamics were
similar in these 2 groups (Table
1), and thus the differences listed below
were interpreted as resulting from the surgical
trauma-induced inflammatory reaction, hydrocortisone treatment, or
both, rather than from sampling errors.
Hydrocortisone reduced surgery-induced slow rolling of leukocytes Cataract surgery induced a strong inflammatory reaction, characterized by an 8-fold increase in the number of rolling leukocytes, that is, 6.1 ± 3.4 rolling leukocytes per minute (mean ± SD) were observed in the conjunctival venules in the specimens taken before the inflammation, whereas up to 46 ± 9.9 rolling leukocytes per minute were observed in them during inflammation (P = .012, Wilcoxon signed rank test, pooled specimens with and without hydrocortisone treatment; Figures 1A and 2A-B). Essentially all slowly rolling cells that could be identified in still video images by their nuclear morphology were granulocytes.
Further analysis of the distribution of velocities of individual rolling leukocytes conducted in the specimens taken during the inflammation showed that the velocity of rolling leukocytes in the conjunctival venules of the nontreated patients was 22 ± 49 µm/s (mean ± SD) (Figure 1B); in hydrocortisone-treated individuals, velocity was significantly elevated, that is, 55 ± 63 µm/s (P = .001 with Mann-Whitney U test). In the specimens taken during the inflammation of hydrocortisone-treated versus nontreated patients, the proportion of adherent or very slowly rolling cells, in particular (range, 0 to 20 µm/s), was significantly reduced (P = .007, with Mann-Whitney U test). Hydrocortisone treatment reduced leukocyte emigration into the tissue The number of extravasated leukocytes was analyzed from adjacent conjunctival tissues by means of electronic video images as well as conventional histological conjunctival biopsies taken after the video microscopy from the same area where the microscopy was performed. Essentially no leukocytes were present in the conjunctival tissue in specimens taken before the inflammation with or without hydrocortisone treatment. In fact, surgery induced a significant elevation in the number of tissue-emigrated cells in nontreated patients (0 ± 0 versus 1480 ± 973; P = .005 with Wilcoxon signed rank test; Figures 1C and 2C). Intravenous preoperative hydrocortisone treatment significantly reduced the number of tissue-infiltrating leukocytes from 1480 ± 973 cells per square millimeter to 714 ± 374 cells per square millimeter (P = .013, Mann-Whitney U test; Figure 1C). All cells within conjunctival tissue that could be identified by their nuclear morphology in still video images were granulocytes. Just as in the in silico histology taken with the confocal video microscope, classical histology provided similar signs of conjunctival inflammation characterized by a large number of tissue-infiltrating leukocytes (Figure 2D).
Induced endothelial expression of P-selectin and ligands for L-selectin on vessels at sites of inflammation To help elucidate the molecular mechanisms of these observations, we took conjunctival biopsies for immunohistological analysis. The presence of endothelial P- and E-selectin, sulfo-sLex glycans (detected with mAbs MECA-79 and 2F3) known to decorate L-selectin ligands, as well as ICAM-1 and VCAM-1 were analyzed immunohistochemically. Of the small conjunctival venules, 15% were P-selectin-positive in biopsies taken before the inflammation with or without hydrocortisone treatment. The proportion of P-selectin-reactive venules was significantly elevated after surgery in the nontreated patients' specimens taken during compared with specimens taken before the inflammation (55% ± 19%; P < .05, Wilcoxon signed rank test; Figure 3A-D). Concomitantly, in the specimens taken during the inflammation, significantly greater reduction occurred in the percentage of venules reactive with endothelial P-selectin in hydrocortisone-treated than in nontreated patients (55% ± 19% versus 15% ± 10%; P = .034, Mann-Whitney U test; Figure 3A).
Another key endothelial adhesion molecule participating in the leukocyte rolling and extravasation, E-selectin, was expressed neither in specimens taken before nor during inflammation. We could induce a strong and specific E-selectin staining in a skin biopsy used as a positive control treated with LPS for 4 hours in vitro, indicating that the negative results in conjunctival biopsies were reliable (data not shown). The sulfated glycans (extended core 1 mucin-type O-glycan)12 detected by MECA-79 were essentially absent from the endothelium of normal conjunctival venules, but were significantly and strongly elevated with inflammation in a small proportion of vessels (8%; P < .05 compared with specimens taken before the inflammation with Wilcoxon signed rank test). Similarly, the sLex-type glycans decorating L-selectin ligands on the endothelium were weakly expressed in a small proportion of normal conjunctival venules, but they were significantly induced with inflammation as detected by mAb 2F3 (9% in specimens taken before the inflammation versus 21% specimens taken during the inflammation; P = .042, Wilcoxon signed rank test). Hydrocortisone treatment, however, had no significant impact on the expression of either MECA-79 (Figure 3E-H) or sLex epitopes (Figure 3I-L). These results suggest that L-selectin ligands were induced in a subset of vessels, but that hydrocortisone pretreatment had no significant effect on them. Analysis of the expression of ICAM-1 and VCAM-1 showed neither ICAM-1 nor VCAM-1 to be present in the vessels in conjunctival biopsies, indicating that leukocyte trafficking in a sterile surgical inflammation model might be dependent on interactions between P- and L-selectin and their ligands but is not dependent on E-selectin, ICAM-1, or VCAM-1.
We show in this study that surgery induced a strong and rapid granulocyte-dominated inflammation, characterized by strong induction of leukocyte rolling on conjunctival venules, which we monitored in real-time noninvasive confocal microscopy. Intravenous hydrocortisone may down-regulate the number of tissue-infiltrating cells, most likely by preventing the adherence of slowly rolling cells to the inner walls of the conjunctival venules. Furthermore, immunohistological biopsies excised from the same conjunctival sites suggest that at least endothelial P-selectin and ligands for L-selectin, but not E-selectin, may participate in the rapid up-regulation of the surgery-induced leukocyte rolling and that the hydrocortisone effect of preventing the slow rolling of leukocytes could be partially mediated by down-regulation of endothelial P-selectin expression in vivo. Corticosteroids inhibit arteriolar leukocyte rolling and their transmigration through the vascular wall in the postcapillary venules,13,14 and in rodent models E-selectin is crucial in controlling the rolling velocities of the leukocytes.15 The surgery-induced inflammation in one rodent model was dependent on P- and L-selectin, but not on E-selectin expression.16 Contrary to this, in a recent human study, incision of the skin under aseptic conditions did not change the levels of endothelial P-selectin, ICAM-1, or VCAM-1, but induced E-selectin expression. These findings may indicate either that different organs respond in an independent manner, that there is variation between species with regard to these responses, or that both are the case. During the past decade, these selectin-mediated functional interactions
have been studied extensively in animal models. Genetic knockout
mutations of several combinations of the selectins (P-, E-, and
L-selectin)17 or 1 or 2 Our data obtained from immunohistological analysis of the conjunctival biopsies suggest that induction of endothelial P-selectin and ligands for L-selectin, but not E-selectin, ICAM-1, or VCAM-1, might be involved in the surgery-induced inflammation, which caused a rapid increase in rolling leukocytes, and especially in the large proportion of adherent and very slowly rolling leukocytes at sites of inflammation in our surgical patients without corticosteroids. Furthermore, the hydrocortisone treatment prevented the slow rolling and was shown to take place concomitantly with the down-regulation of endothelial P-selectin expression. Taken together, these results suggests that current anti-inflammatory drugs may also have a potential influence on leukocyte trafficking in vivo. Finally, we conclude that direct noninvasive monitoring of inflammatory reactions in human patients can be carried out in medically relevant inflammatory models.
We thank Eeva Linnolahti, Esko Järvinen, Veli-Pekka Suomalainen, and Heikki Saaren-Seppälä for providing histological biopsies and for smooth collaboration, and Minna Vesaluoma for aid with statistics.
Submitted April 3, 2002; accepted May 13, 2002.
Prepublished online as Blood First Edition Paper, May 24, 2002; DOI 10.1182/blood-2002-04-1017.
Supported by grants from the Academy of Finland (R.R.), Technology Development Center of Finland (R.R.), and the Sigrid Juselius Foundation (R.R. and T.M.T.T.); the Rector's grant, Helsinki University (T.M.T.T.); research funds from the Helsinki University Central Hospital (T.M.T.T., R.R., and T.P.); and the Eye Foundation (M.H. and J.A.O.M.).
M.H. and J.A.O.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Risto Renkonen, Rational Drug Design Program, Biomedicum and Haartman Institute, PO Box 63, FIN-00014 University of Helsinki, Finland; e-mail: risto.renkonen{at}helsinki.fi.
1. Barnes PJ. Anti-inflammatory actions of glucocorticosteroids: molecular mechanisms. Clin Sci. 1998;94:557-571[Medline] [Order article via Infotrieve]. 2. Barnes PJ. Corticosteroids, IgE, and atopy. J Clin Invest. 2001;107:265-266[Medline] [Order article via Infotrieve]. 3. Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol. 1999;72:209-253[Medline] [Order article via Infotrieve]. 4. Lowe JB. Glycosylation, immunity, and autoimmunity. Cell. 2001;104:809-812[CrossRef][Medline] [Order article via Infotrieve].
5.
Rosen SD.
Endothelial ligands for L-selectin: from lymphocyte recirculation to allograft rejection.
Am J Pathol.
1999;155:1013-1020
6.
Vestweber D, Blanks JE.
Mechanisms that regulate the function of the selectins and their ligands.
Physiol Rev.
1999;79:181-213 7. Kirveskari J, Vesaluoma MH, Moilanen JA, et al. A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients. Nat Med. 2001;7:376-379[CrossRef][Medline] [Order article via Infotrieve]. 8. Li HF, Petroll WM, Moller-Pedersen T, Maurer JK, Cavanagh HD, Jester JV. Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF). Curr Eye Res 1997;16:214-221[CrossRef][Medline] [Order article via Infotrieve]. 9. Petroll WM, Jester JV, Cavanagh HD. Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: system design and calibration. Scanning. 1996;18:45-49[Medline] [Order article via Infotrieve].
10.
Mitsuoka C, Sawada-Kasugai M, Ando-Furui K, et al.
Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.
J Biol Chem
1998;273:11225-11233 11. Michie SA, Streeter PR, Bolt PA, Butcher EC, Picker LJ. The human peripheral lymph node vascular addressin: an inducible endothelial antigen involved in lymphocyte homing. Am J Pathol. 1993;143:1688-1698[Abstract]. 12. Yeh JC, Hiraoka N, Petryniak B, et al. Novel sulfated lymphocyte homing receptors and their control by a core1 extension beta 1,3-N-acetylglucosaminyltransferase. Cell. 2001;105:957-969[CrossRef][Medline] [Order article via Infotrieve].
13.
Zhang XW, Thorlacius H.
Dexamethasone inhibits arteriolar leukocyte rolling and adhesion by tumor necrosis factor- 14. Mancuso F, Flower RJ, Perretti M. Leukocyte transmigration, but not rolling or adhesion, is selectively inhibited by dexamethasone in the hamster post-capillary venule: involvement of endogenous lipocortin 1. J Immunol. 1995;155:377-386[Abstract].
15.
Kunkel EJ, Ley K.
Distinct phenotype of E-selectin-deficient mice: E-selectin is required for slow leukocyte rolling in vivo.
Circ Res.
1996;79:1196-1204
16.
Kunkel EJ, Jung U, Bullard DC, et al.
Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1.
J Exp Med.
1996;183:57-65
17.
Jung U, Ley K.
Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin.
J Immunol.
1999;162:6755-6762 18. Maly P, Thall AD, Petryniak B, et al. The alpha(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficing through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell. 1996;86:643-653[CrossRef][Medline] [Order article via Infotrieve]. 19. Homeister JW, Thall AD, Petryniak B, et al. The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing. Immunity. 2001;15:115-126[CrossRef][Medline] [Order article via Infotrieve].
20.
Oriol R, Mollicone R, Cailleau A, Balanzino L, Breton C.
Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria.
Glycobiology.
1999;9:323-334
21.
Satomaa T, Renkonen O, Helin J, Kirveskari J, Mäkitie A, Renkonen R.
O-glycans on human high endothelial CD34 putatively participating in L-selectin recognition.
Blood.
2002;99:2609-2611
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. K. Toppila-Salmi, J. P. Myller, T. V. M. Torkkeli, J. V. Muhonen, J. A. Renkonen, M. E. Rautiainen, and R. L. O. Renkonen Endothelial L-Selectin Ligands in Sinus Mucosa during Chronic Maxillary Rhinosinusitis Am. J. Respir. Crit. Care Med., June 15, 2005; 171(12): 1350 - 1357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Helinto, R. Renkonen, T. Tervo, M. Vesaluoma, H. Saaren-Seppala, T. Haahtela, and J. Kirveskari Direct In Vivo Monitoring of Acute Allergic Reactions in Human Conjunctiva J. Immunol., March 1, 2004; 172(5): 3235 - 3242. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
