Departments of 3 Molecular and Cellular Physiology and 2 Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, Louisiana 71130; and 1 First Department of Surgery, Nagoya University School of Medicine, Nagoya 466-8550, Japan
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ABSTRACT |
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Quantitative estimates of endothelial cell adhesion molecule expression have revealed that some adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] are abundantly expressed in different vascular beds under normal conditions. The objective of this study was to determine whether the enteric microflora contribute to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules in the gastrointestinal tract and other regional vascular beds. The dual radiolabeled monoclonal antibody technique was used to measure endothelial expression of ICAM-1, ICAM-2, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in conventional, germ-free mice and germ-free mice receiving the cecal contents of conventional mice to reestablish the enteric microflora (total association). Constitutive ICAM-1 expression was significantly lower in the splanchnic organs (pancreas, stomach, small and large intestine, mesentery, and liver), kidneys, skeletal muscle, and skin of germ-free mice compared with their conventional counterparts. These differences were abolished after total association of germ-free mice with the indigenous gastrointestinal flora. The expression of ICAM-2, VCAM-1, and E-selectin in the various tissues studied did not differ between conventional and germ-free mice. These findings indicate that the indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extraintestinal tissues.
germ-free mice; leukocyte-endothelial cell adhesion; inflammation; bacterial translocation; host defense
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INTRODUCTION |
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EPITHELIAL CELLS of the digestive tract, airways, and skin are normally exposed to large numbers of microorganisms. The indigenous microflora found in these tissues can serve important physiological functions, such as vitamin K biosynthesis, ammonia production, and host defense against colonization and/or translocation by certain pathogenic bacteria (5, 37). However, these microorganisms may also render tissues that are normally exposed to the external environment more vulnerable to certain pathological processes. For example, there is a large body of circumstantial evidence that links the pathogenesis of inflammatory bowel disease (IBD) to the indigenous gastrointestinal (GI) microflora (36, 37). Some experimental models of IBD gene-targeted mice [interleukin (IL)-2, IL-10 deficient, and HLA-B27 transgenic] do not develop inflammation under specific pathogen-free conditions (20, 34). Although the role of enteric bacteria in initiating and/or exacerbating acute and chronic intestinal inflammation has been extensively studied, the mechanisms that explain the linkage between the gut microflora and the recruitment and activation of inflammatory cells in intestinal tissue remain poorly defined.
The development of an inflammatory response involves highly coordinated and sequential adhesive interactions between leukocytes and endothelial cells that are manifested as leukocyte rolling, adherence, and emigration (30). Each of these steps in the recruitment of leukocytes is mediated by unique adhesion glycoproteins that are expressed on the surface of activated leukocytes or endothelial cells. Some of the adhesion molecules produced by endothelial cells are expressed on the cell surface under normal physiological circumstances. For example, large amounts of intercellular adhesion molecule (ICAM)-1 and ICAM-2 are expressed on the surface of endothelial cells in most regional vascular beds (13, 22, 31), whereas P-selectin is constitutively expressed in only a few vascular beds (9). Other adhesion molecules, such as E-selectin, are not normally expressed on endothelial cells, but their expression can be transcriptionally induced by cytokines, bacterial endotoxins, and other mediators (9). Although it is well known that the basal (constitutive) expression of ICAM-1 is normally high and that its expression can be increased in a transcription-dependent manner by cytokines and bacterial toxins (30), most studies of ICAM-1 expression have focused on the factors that regulate its induction, such as nuclear transcription factors. Much less attention has been devoted to defining what regulates the constitutive expression of this adhesion molecule. The importance of constitutively expressed ICAM-1 in mounting an inflammatory response is evidenced by reports (15, 21, 44) describing ICAM-1-dependent leukocyte-endothelial cell adhesion that occurs in the absence of an increased ICAM-1 expression.
There is some circumstantial evidence in the literature that supports the possibility that indigenous GI bacteria may contribute to the regulation of constitutive and induced ICAM-1 expression in the intestinal vasculature. We (3) have previously reported that pretreatment of rats with the antibiotic metronidazole significantly reduces leukotriene B4 (LTB4)-induced, ICAM-1 dependent leukocyte-endothelial cell adhesion in mesenteric venules. In a separate study (18), it was shown that oral administration of antibiotics (kanamycin and/or metronidazole) for 2 days significantly reduces the constitutive expression of ICAM-1 in certain tissues (e.g., the cecum). Although the antibiotics were administered in an attempt to reduce the total number of viable enteric bacteria, interpretation of these findings was complicated by the fact that metronidazole exerts antioxidant properties (2) and the possibility that these antibiotics alter the normal ecological equilibrium in the GI tract, thereby promoting microbial translocation from gut lumen into extravascular and vascular compartments (6). To directly address the contribution of indigenous GI bacteria to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules (CAMs), we quantified the expression of these adhesion molecules in different vascular beds of conventional and germ-free mice, thereby negating the need to administer antibiotics with potential nonspecific properties that could also influence endothelial CAM expression.
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METHODS |
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Animals. Male BALB/c conventional and germ-free mice, 5-9 wk of age, were obtained from Taconic Farm (Germantown, NY) and maintained in a sterile environment. All mice were maintained on steam-sterilized, standard mouse chow until the experiment. The experimental procedures described herein were reviewed and approved by the Louisiana State University Health Sciences Center-Shreveport Institutional Animal Care and Use Committee and performed according to the criteria outlined in the National Institutes of Health guidelines.
Monoclonal antibodies. The binding monoclonal antibodies (MAbs) used for the in vivo assessment of ICAM-1, VCAM-1, ICAM-2, and E-selectin expression were as follows: YN1, a rat IgG2b against mouse ICAM-1 (11, 14, 30); MK1.9.1., rat IgG1 targeted against mouse VCAM-1 (both YN1 and MK1.9.1 were provided by Dr Mary Gerritsen from Bayer, West Haven, CT) (13, 24, 25); 10E6, a rat IgG2 against mouse E-selectin (9, 28); and 3C4, a rat IgG2a that reacts with mouse ICAM-2 (Pharmingen). P23, a nonbinding murine IgG1 directed against human P-selectin (Pharmcia-Upjohn, Kalamazoo, MI), was also used in the experimental protocol (23).
Radioiodination of MAbs. All of the binding MAbs, YN1, MK1.9.1, 3C4, and 10E6, were labeled with 125I (Du Pont NEN, Boston, MA), while the nonbinding MAb (P23) was labeled with 131I. Radioiodination of the MAbs was performed by the iodogen method (10). Briefly, 250 µg of protein were incubated with 250 µCi of Na 125I and 125 µg of iodogen at 4°C for 12 min. The radiolabeled MAbs were separated from free 125I by gel filtration on a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden). The column was equilibrated with phosphate buffer containing 1% BSA and was eluted with the same buffer. Two fractions of 2.5 ml each were collected, the second of which contained the labeled antibody. The absence of free 125I or 131I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. This technique has been used previously for preparation of both anti-rat and anti-mouse CAM-binding MAbs (9, 13, 31).
Animal procedures. The mice were anesthetized with ketamine hydrochloride (150 mg/kg body wt im) and xylazine (10 mg/kg body wt im). The right carotid artery and the left jugular vein were cannulated with polyethylene tubing. To measure ICAM-1 expression, a mixture of 10 µg of anti-ICAM-1 MAb (YN1) and a dose of unlabeled anti-ICAM-1 MAb (5 µg) with an amount of 131I necessary to ensure a total 131I-injected activity of 400,000-600,000 counts/min (cpm) was administered through the jugular vein cannula (total vol, 200 µl). In the VCAM-1 expression experiments, 10 µg of 125I-anti-VCAM-1 MAb (MK1.9.1) and an appropriate amount of 131I-P23 (400,000-600,000 cpm) were administered through the jugular vein cannula (total vol, 200 µl). In some experiments, estimates of tissue endothelial surface area were obtained by quantifying the expression of ICAM-2, which has been shown to be refractory to cytokine or endotoxin stimulation in normal vascular beds (22, 27). ICAM-2 expression was determined by a mixture of 10 µg of 125I- anti-ICAM-2 MAb and 20 µg of unlabeled anti-ICAM-2 MAb with an appropriate amount of 131I-P23 (400,000-600,000 cpm) administered through the jugular vein cannula (total vol, 200 µl). E-selectin expression, which is undetectable in most of the organs except for the skin under basal conditions (16, 22), was measured to determine whether the indigenous GI microflora contribute to the expression of this adhesion molecule in cutaneous tissue. A mixture of 5 µg of 125I- anti-E-selectin MAb (10E6) with an appropriate amount of 131I-P23 (400,000-600,000 cpm) was given through the jugular vein cannula (total vol, 200 µl). These doses of binding MAbs were chosen based on pilot studies demonstrating optimum activity and receptor saturation in the tissues examined under basal conditions.
A blood sample was obtained through the carotid artery catheter at 5 min after injection of the MAb mixture. Thereafter, the animals were heparinized (1 mg/kg sodium heparin) and rapidly exsanguinated by vascular perfusion with sodium phosphate buffer via the jugular vein and simultaneous blood withdrawal via the carotid artery. The inferior vena cava was then severed at the thoracic level, and the carotid artery was perfused with sodium phosphate buffer. After completion of the exchange transfusion, organs were harvested and weighed.Calculation of CAM expression
125I (binding MAb) and 131I (nonbinding MAb)
activities in different organs and in 50-µl aliquots of cell-free
plasma were counted in a 14800 Wizard 3 gamma counter (Wallac, Turku,
Finland), with automatic correction for background activity and
spillover. The injected activity in each experiment was calculated by
counting a 2-µl sample of the mixture containing the radiolabeled
MAbs. The radioactivities remaining in the tube used to mix the MAbs, the syringe used to inject the mixture, and the jugular vein catheter were subtracted from the total calculated injected activity. The accumulated activity of each MAb in an organ was expressed as the
percentage of the injected dose (%ID) per gram of tissue. The formula
used to calculate CAM expression was as follows: CAM expression = (%ID/g for 125I) (%ID/g for
131I) × (%ID 125I in plasma)/(% ID
131I in plasma). This formula was modified from the
original method (31) to correct the tissue accumulation of
nonbinding MAb for the relative plasma levels of both binding and
nonbinding MAbs (17). This value (%ID) was converted to
micrograms of MAb per gram of tissue by multiplying the above value by
the total injected binding MAb (in µg), divided by 100.
Experimental protocols. CAM expression was determined in the organs of germ-free mice and conventional mice under control (constitutive) conditions. In another series of experiments, germ-free mice were inoculated intragastrically with a cecal homogenate from conventional mice and then housed under unsterilized, conventional conditions (total association of microflora) (6) to ensure that the altered CAM expression in germ-free mice could be restored to normal levels when reconstituted with the indigenous GI microflora. ICAM-1 expression was measured 2 and 4 wk after the association, when immunologic indexes in germ-free animals are known to be restored to normal levels (26, 40, 43).
Statistics. The data were analyzed using ANOVA with the Scheffé (post hoc) test. Either paired or unpaired Student's t-test was used when only two groups were compared. All values are presented as means ± SD. Statistical significance was set at P < 0.05.
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RESULTS |
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Figure 1 compares the constitutive
expression of different endothelial CAMs (ICAM-1, VCAM-1, and
E-selectin) in the lung, liver, ileum, and skin of conventional and
germ-free mice. Table 1 summarizes the
data obtained for ICAM-1, VCAM-1, and ICAM-2 for all of the organs
studied. These data indicate that constitutive ICAM-1 expression is
significantly lower in the GI tract, liver, pancreas, kidneys, skeletal
muscle, and skin of germ-free mice than in their conventional
counterparts. ICAM-1 expression in the lung, heart, and brain did not
differ between the two groups of animals. No significant differences in
the expression of VCAM-1 or E-selectin were noted for any tissue in
these animals. ICAM-2 expression also did not differ between tissues of
germ-free and conventional mice except for the skin, which exhibited a
lower ICAM-2 expression in germ-free mice, compared with conventional mice.
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Figure 2 demonstrates that intragastric
inoculation of germ-free mice with cecal microflora from conventional
mice restored ICAM-1 expression to normal (conventional) values in the
liver, ileum, and skin. Although 2 wk of bacterial association were
required to restore ICAM-1 expression to conventional values in the
liver and ileum, 4 wk of association were needed to fully restore
ICAM-1 expression in the skin. ICAM-1 expression in the lung, heart, and brain, which was not reduced in germ-free compared with normal mice, was also unaffected by bacterial association.
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DISCUSSION |
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The influence of bacteria and their products on the immune system has been extensively studied. It is generally well recognized that bacterial products are potent activators of different arms of the immune system and that, as a consequence of this activation, leukocytes are recruited to sites of bacterial invasion. A similar involvement of bacterial products has been invoked to explain the large resident population of phagocytic cells found in certain tissues that are normally exposed to a high density of indigenous bacteria. The fact that the intestine normally exists in a state of controlled inflammation has been attributed to both the quality and quantity of microorganisms that normally reside in the gut lumen (7). While it is often assumed that the presence of enteric bacteria influences the environment within the intestinal mucosa in a manner that favors the recruitment of phagocytic cells, the details regarding the nature of this proinflammatory phenotype of gut tissue remain poorly defined. Hence, in this study we addressed the possibility that the indigenous GI microflora in healthy animals exert a regulatory influence on the basal expression of endothelial CAMs in the GI tract and in other regional vascular beds.
The overall objective of this study was achieved by comparing endothelial CAM expression in different vascular beds of conventional and germ-free mice. The dual radiolabeled MAb technique, which detects the relative accumulation of adhesion molecule-specific binding and nonbinding MAbs in the vasculature (31), was used to quantify endothelial CAM expression. This technique has been previously employed to measure endothelial CAM expression in regional vascular beds of normal animals and in animal models of portal hypertension (32), arterial hypertension (19), intestinal stasis (18), ischemia and reperfusion (14), and IBD (8). The present study adds to the growing body of literature on endothelial CAM regulation by demonstrating a significant attenuation of constitutive ICAM-1 expression in certain tissues of germ-free mice relative to conventional mice. The dependence of this response on the reduced indigenous bacterial population is supported by our finding that constitutive ICAM-1 expression in germ-free mice is restored to normal levels when germ-free mice are reconstituted with indigenous cecal microflora.
An important issue raised by our findings is whether a specific constituent of the gut microflora is responsible for the elevated ICAM-1 expression in tissues of conventional mice compared with germ-free mice. One potential approach for addressing this issue would involve selective reconstitution of the microflora in germ-free mice. Because there are 400-500 species of bacteria that normally reside in the GI tract of humans and mice (42), a rather exhaustive analysis would be required to determine which bacterial species within the indigenous GI microflora mediate the altered ICAM-1 expression in germ-free mice. Furthermore, candidate bacterial species that could mediate the observed differences between germ-free and conventional mice are made less apparent by our (18) previously published observation that constitutive ICAM-1 expression in the cecum of conventional mice is reduced to a comparable extent by kanamycin, metronidazole, or a combination of the aerobic and anaerobic antibiotics.
An interesting and potentially important observation in this study is
the tissue specificity of the ICAM-1 responses in germ-free mice
compared with conventional mice. Significant reductions in ICAM-1
expression were noted in some (intestinal and skin) but not all (e.g.,
lung) tissues that are normally exposed to indigenous bacteria.
Similarly, some (the brain and heart) but not all (the liver, pancreas,
mesentery, kidneys, and skeletal muscle) organs that are not normally
in direct contact with bacteria exhibited no difference in ICAM-1
expression between germ-free and conventional mice. These
tissue-specific responses, particularly those that appear unrelated to
direct bacterial exposure, suggest that some systemic factor mediates
the bacteria-dependent elevation in constitutive ICAM-1 expression
observed in certain tissues and that differences in endothelial cell
(or macrophage) responsiveness to this factor(s) account for the tissue
specificity. Although it is tempting to speculate that bacterial
endotoxin may be the circulating factor that mediates these
tissue-specific responses, there is much evidence in the literature
that argues against this possibility. For example, all of the tissues
examined in this study respond to exogenous endotoxin with large
increases in the expression of ICAM-1 (4, 31). While a similar argument can be made against the
involvement of some cytokines, such as tumor necrosis factor-
(TNF-
) (13, 17), it is possible that
certain cytokines are produced in response to the indigenous bacterial
load that can account for the observed pattern of ICAM-1 expression.
Another interesting observation in this study is the fact that ICAM-1
was largely the only endothelial CAM that was affected by the germ-free
condition, i.e., the expression of other adhesion molecules (ICAM-2,
VCAM-1, and E-selectin) was virtually unaffected by the absence of the
resident bacterial population. The absence of a difference in VCAM-1
and E-selectin expression between germ-free and conventional mice is
particularly interesting in view of reports (30,
39) showing that ICAM-1, VCAM-1, and E-selectin respond in
a similar fashion to bacterial endotoxin, TNF-, and IL-1. These
shared responses to endotoxin and cytokine challenge are explained in
part by a shared dependence of the genetic responses to certain nuclear
transcription factors, such as nuclear factor-
B (NK-
B) and
activating protein-1 (AP-1) (1, 35). Hence,
it would appear unlikely that endotoxin, TNF-
, IL-1, or other
factors that promote ICAM-1 expression through AP-1 or NF-
B
activation could account for the differences in constitutive ICAM-1
expression observed between germ-free and conventional mice. However,
dissociated patterns of gene expression and/or turnover between ICAM-1
and VCAM-1/E-selectin have been recently observed during attempts to
specifically inhibit the actions of transcription factors, including
NF-
B and AP-1, or to modify intracellular levels of cAMP or GTP
(1, 11, 12, 29,
41). This suggests that the ICAM-1 gene is regulated via a
different combination of signal transduction pathways.
A well-known feature of the cutaneous circulation is its significant expression of E-selectin on endothelial cells under basal conditions. This property of cutaneous endothelial cells has been demonstrated on cultured endothelial cells (16) and in vivo, using immunohistochemical methods as well as radiolabeled MAbs (22, 33). Skin homing receptors, such as cutaneous lymphocyte-associated antigen, expressed by certain T lymphocyte populations are known to bind with high affinity to cutaneous E-selectin (33). Consequently, it has been proposed that the high basal expression of E-selectin in cutaneous microvessels may represent an important mechanism for immune surveillance in this tissue. In the present study, we found no difference in the levels of constitutive E-selectin expression in the skin of germ-free and conventional mice. This suggests that the bacteria normally found on the skin surface do not elicit the uniquely high endothelial cell expression of this adhesion molecule.
In conclusion, the results of this study indicate that indigenous bacteria elaborate a signal that accounts for a significant fraction of constitutive ICAM-1 expression in the GI tract, liver, skin, and other tissues. The constitutive expression of other endothelial CAMs, such as E-selectin, VCAM-1, and ICAM-2, appears to be unaffected by the normal microflora. These findings may have important implications in the selective trafficking of circulating leukocytes to specific vascular beds under normal physiological conditions.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant P01 DK-43785 and by a Grant-in-Aid (C 11671230) from the Ministry of Education, Science, Sports, and Culture of Japan (Y. Nimura).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D.N. Granger, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Medical Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 (E-mail: dgrang{at}lsumc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 14 September 1999; accepted in final form 17 February 2000.
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