There is emerging evidence for a role of the CD40/CD40 ligand (CD40L) dyad as a signaling mechanism in different inflammatory conditions. The aims of this study were to 1) quantify the constitutive and induced expression of CD40 in different regional vascular beds of the mouse and 2) assess the role of CD40L as a modulator of vascular endothelial CD40 expression. The dual radiolabeled monoclonal antibody technique was used to quantify the expression of endothelial CD40 in control and LPS-challenged wild-type (WT) mice. Significant constitutive CD40 expression was detected in several vascular beds of WT mice with lung, kidney, and small intestine exhibiting the highest expression, whereas the liver and stomach showed no detectable baseline expression. LPS administration elicited two- to sevenfold increases in CD40 expression in several tissues (heart, kidney, and intestine) within 4 h, whereas other organs (brain) required up to 48 h to exhibit CD40 upregulation. CD40 expression was not detected in unstimulated or LPS-challenged CD40−/− mice. Constitutive expression of CD40 was profoundly reduced in unstimulated CD40L−/− mice, but the LPS-induced CD40 upregulation did not differ between CD40L−/− and WT mice. Depletion of platelets or T lymphocytes, the major CD40L-expressing cells in blood, also resulted in a profound reduction in basal CD40 expression. These findings demonstrate significant endothelial expression of CD40 under basal conditions in different vascular beds and increased CD40 expression after endothelial cell activation with LPS. Platelet- and T-lymphocyte-associated CD40L appears to play a major role in regulating the density of CD40 expression on vascular endothelial cells in vivo.
- CD40 ligand
- dual radiolabeling
cd40-cd40 ligand (CD40L) interactions are known to play an important role in adaptive immune responses, inflammation, and hemostasis and coagulation (16, 17). CD40, a member of the TNF receptor family, is widely distributed, primarily on cells of the vasculature where it is constitutively expressed on endothelial cells. The ligand for CD40 is CD154 (or CD40L), another transmembrane protein that is structurally related to the cytokine TNF (37). CD40L is found on cells of the immune system (e.g., T lymphocytes and mast cells) and on activated platelets. Platelets can express 600–1,000 copies of CD40L within seconds of activation and account for >90% of all CD40L (2). Engagement of CD40L on activated, adherent platelets (or T lymphocytes) with constitutively expressed CD40 on endothelial cells results in phenotypic changes in the endothelial cell that are similar to that induced by TNF-α, i.e., increased expression of E-selectin, ICAM-1, and VCAM-1 and an increased secretion of the chemokines, IL-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1) (19, 20). Hence, the interaction of CD40 with its ligand (CD40L) causes vascular endothelial cells to assume a proinflammatory and prothrombogenic phenotype, as evidenced by reports describing an attenuation of leukocyte and platelet recruitment in the microvasculature of CD40−/− or CD40L−/− mice subjected to an inflammatory insult, such as ischemic stroke (24).
Although it is widely recognized that CD40 is expressed on vascular endothelial cells, relatively little is known about the magnitude of CD40 expression on endothelial cells in different organs, the in vivo kinetics of CD40 expression after endothelial cell activation, and the factors that regulate CD40 expression in situ. Immunohistochemical studies have revealed that CD40 is basally expressed on endothelial cells in most tissues (34). However, this detection method does not afford the resolution necessary to address whether the intensity of basal CD40 expression on endothelial cells differs among tissues or whether certain tissues have a greater capacity to upregulate CD40 after endothelial cell activation. Cultured human umbilical vein endothelial cells (HUVEC) have been shown to express a low level of CD40 antigen under basal conditions, but these cells exhibit a 1.3- to 8-fold increase in CD40 expression after exposure to interferon-γ and/or TNF-α (25, 39). The increased expression of CD40 on cultured HUVEC occurs within 8 h but continues to rise for as long as 72 h after cytokine challenge (25). Whether the magnitude and kinetics of CD40 expression that is observed on activated HUVEC are representative of the changes that occur in the microvasculature of different organs remains unclear. Differences between CD40 expression on HUVEC and microvascular endothelial cells in various organs appear likely in view of previous studies of endothelial cell adhesion molecule (CAM) expression after systemic challenge with bacterial endotoxin (LPS) or cytokines that revealed substantial organ-to-organ differences in intensity and time course of CAM expression that were not predicted by cultured HUVEC (12).
Previous efforts to evaluate the responses of cultured HUVEC to CD40-CD40L engagement have revealed that although endothelial cells do not normally express CD40L, activation of HUVEC with CD40L-positive myeloma cells induces the synthesis and expression of CD40L on the endothelial cell surface (38). This CD40L-dependent expression of CD40L on HUVEC is also seen when HUVEC are exposed to CD40L-positive platelets (7). These observations suggest that endothelial cells can amplify the inflammatory response to CD40-CD40L interaction by promoting the expression of CD40L on endothelial cells, which allows the vascular cells to also interact with CD40 of the same population of blood cells or with other inflammatory cells that express CD40. These interesting observations raise the possibility that endothelial cells may also respond to CD40L engagement by increasing the synthesis and surface expression of CD40, which might be expected in view of the ability of TNF-α to induce CD40 expression on HUVEC (25). Whether CD40L contributes to the regulation of CD40 expression on endothelial cells has not been previously addressed.
In the present study, endothelial expression of CD40 was quantified by using the dual radiolabeled MAb technique. This technique, which utilizes radiolabeled MAbs that are directed against specific proteins (e.g., adhesion molecules and receptors) expressed on the surface of endothelial cells, allows for the detection of organ-to-organ differences in constitutive and induced expression of the endothelial cell-associated proteins with a precision not previously possible when using immunohistochemical procedures (31). Herein, the technique was used to 1) determine the magnitude of basal CD40 expression on endothelial cells in different organs of the mouse, 2) define the in vivo kinetics of CD40 expression after systemic endothelial cell activation with bacterial endotoxin LPS, and 3) assess the role of CD40L as a modulator of CD40 expression on vascular endothelial cells.
MATERIALS AND METHODS
The animals used in the experiments were 6- to 8-wk-old male C57BL/6J mice [wild-type (WT) control strain], CD40 knockout (CD40−/−), CD40L−/−, and recombinase-activating gene (RAG)-1−/− mice (all developed on a C57BL/6J background), obtained from Jackson Laboratory (Bar Harbor, ME). Animals were housed under specific pathogen-free conditions in standard cages and were fed standard laboratory chow and water ad libidum until the desired age. The experimental procedures were performed according to the criteria outlined in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee.
The MAbs used for in vivo assessment of CD40 and platelet endothelial CAM-1 (PECAM-1) expression were 3/23, a purified binding rat immunoglobulin (IgG2a) that is specific for mouse CD40, MEC 13.3, a purified binding rat immunoglobulin (IgG2a) that is directed against mouse PECAM-1 (both from BD Pharmingen, San Diego, CA), and P-23, a nonspecific, nonbinding murine IgG1 directed against human P-selectin (provided by Dr. Donald C. Anderson, Pharmacia-Upjohn, Kalamazoo, MI). The specific binding (3/23 and MEC 13.3) and nonbinding (P-23) MAbs were labeled with 125I and 131I, respectively (DuPont-New England Nuclear, Boston, MA), using the iodogen method as described previously (13, 21).
The mice were anesthetized intramuscularly with 150 mg/kg ketamine and 7.5 mg/kg xylazine. The right jugular vein and right carotid artery were cannulated with polyethylene tubing (PE-10). To measure CD40 or PECAM-1 expression, a mixture of 125I-labeled binding MAb (either 20 μg anti-CD40 or 10 μg anti-PECAM-1) and 0.5–5 μg of nonbinding 131I-labeled MAb (adjusted to ensure a total 131I-injected activity of 500,000 ± 100,000 counts/min) was injected through the jugular vein catheter (total volume 200 μl). Pilot studies utilizing a 20-μg dose of 125I-labeled anti-CD40 MAb in conjunction with the nonbinding MAb showed that this dose provided optimum activity to accurately assess CD40 expression and to ensure receptor saturation under constitutive and stimulated conditions. The dose of PECAM-1 was based on a previous study in Granger's laboratory (21). Blood samples (200 μl) were obtained from the carotid artery catheter 5 min after injection of the MAb mixture for measurement of plasma 125I and 131I activity. Thereafter, an isovolemic blood exchange was rapidly performed by perfusion with 6 ml of bicarbonate-buffered saline (BBS) through the jugular vein catheter with simultaneous blood withdrawal through the carotid artery catheter. This was followed by perfusion of 15 ml of BBS through the carotid artery catheter after severing the inferior vena cava at the thoracic level. The brain, heart, lung, stomach, small intestine, large intestine, liver, kidneys, pancreas, mesentery, eyes, and muscle were harvested. All the organs were weighed before radioactivity measurements were taken (12).
Calculation of CD40 and PECAM-1 expression.
The method for calculating CD40 and PECAM-1 expression has been described previously (12, 21). Briefly, activity of 125I and 131I (marking the binding MAb and the nonbinding MAb, respectively) in the tissue and in 50-μl samples of cell-free plasma was counted in a 14800 Wizard 3 counter (Wallac, Turku, Finland) with automatic correction for background activity and spillover. A 2-μl aliquot of the preinjection mixture of radiolabeled MAbs was assayed to determine total injected activity of each labeled MAb. The tube used to mix the MAbs and the infusion syringe were likewise counted, and their activities were subtracted from the total levels. The accumulated activity of each labeled MAb in each organ was expressed as the percentage of the injected activity per gram tissue. CD40 or PECAM-1 expression was then calculated by subtracting the accumulated activity per gram tissue of the nonbinding MAb from the activity of the binding MAb. This value, expressed as the percent-injected dose per gram tissue, was converted to nanograms MAb per gram tissue by multiplying the above value by the total injected binding MAb. Previous studies (31) have shown that the binding MAbs retain their functional activity after radioiodination.
Four series of experiments were performed.
The first set of experiments compared the vascular distribution of CD40 between different organs under nonstimulated conditions. The dual radiolabeled MAb technique was used to quantify the expression of CD40 and PECAM-1 in different regional vascular beds of WT mice, and the density of CD40 was normalized to the vascular surface area of each organ with the use of PECAM-1 expression as an indicator of the endothelial cell surface area, as previously described (12, 21).
Second, constitutive CD40 expression was measured in WT, CD40−/− (to ensure the CD40 MAb was binding specifically to its receptor), and CD40L−/− mice (to determine whether CD40L was an important factor in the regulation of basal expression of CD40). To assess the contribution of platelet-derived and T-lymphocyte-derived CD40L to the regulation of constitutive CD40 expression, thrombocytopenic animals [anti-platelet serum (APS) group] and lymphocyte-deficient RAG-1−/− animals were used, respectively.
Third, the dual radiolabeled MAb technique was also used to define the magnitude and kinetics of CD40 induction in different regional vascular beds of WT mice after intraperitoneal LPS injection (5 μg in 200 μl normal saline, Escherichia coli LPS, serotype 0111:B4, Sigma Chemical, St. Louis, MO). Animals were euthanized at various time points (2, 4, 8, 12, 24, 48 h) after LPS administration.
Fourth, to determine the contribution of CD40L to the induction of CD40 by LPS, WT, CD40−/−, and CD40L−/− mice were injected with 5 μg LPS and allowed to recover for 4 h before CD40 expression was measured as described above.
Thrombocytopenia was induced in some animals by intraperitoneal injection of 1 mg/kg rabbit anti-mouse APS (Accurate Chemical and Scientific, Westbury, NY) 48 and 24 h before the experiment. Thrombocytopenia was confirmed by manual platelet count of whole blood samples at the end of the experiment and compared with platelet counts performed before APS administration in the same animals.
Standard statistical analyses, i.e., one-way ANOVA with Fisher (post hoc) test, were applied to the data. Statistical significance was set at P < 0.05. All values are means ± SE, except for data in Table 1, which are means (SD). Statistical differences in CD40 expression among different organs in Table 1 were determined by using the paired Student's t-test. For normalization of endothelial CD40 expression to vascular surface area (PECAM-1 expression), it was assumed that the errors of endothelial CD40 and PECAM-1 expression were independent and not correlated. Therefore, the ratio of endothelial CD40-to-PECAM-1 expression was calculated as the ratio of the mean values of these variables, as described previously (11). The SD of the ratio of endothelial CD40-to-PECAM-1 expression was calculated as: (1) where X, Y, and Z are the mean values for PECAM-1, CD40, and the ratio of CD40 to PECAM-1, respectively, and SDCD40 and SDPECAM-1 refer to the SD of CD40 and PECAM-1 values, respectively.
Constitutive expression of CD40 in different vascular beds.
Significant regional differences were found in the tissue distribution of CD40 in the unstimulated state (Table 1) with every organ showing a statistically significant difference (P < 0.05) in the level of CD40 expression compared with at least four other organs tested. The lung showed the highest level of CD40 expression, followed by the kidneys, small intestine, and heart, whereas CD40 expression was not detectable in some tissues, e.g., liver and stomach. Because the binding of CD40 MAb in a vascular bed is determined by both the density of CD40 expression per endothelial cell and endothelial cell surface area, estimates of the latter were obtained by using PECAM-1 expression measurements in the same tissues under unstimulated conditions (Table 1). PECAM-1 expression has been previously shown to provide a valid estimate of endothelial cell surface area (11, 12, 21). A CD40-to-PECAM-1 ratio was calculated to normalize CD40 expression in each tissue to endothelial cell surface area. These estimates revealed that the pulmonary vasculature exhibits the highest density of CD40 expression per endothelial area, and the kidney, brain, and small intestine also expressed high levels.
Constitutive CD40 expression in CD40 and CD40L deficient mice.
To demonstrate the specificity of the 125I-labeled anti-CD40 MAb accumulation in tissues, estimates of CD40 expression were obtained from mice that are genetically deficient in CD40 (CD40−/−). Figure 1 illustrates that there was no accumulation of the anti-CD40 MAb in the heart, kidneys, brain, and small intestine of CD40−/− mice. Additionally, in all other organs, which showed constitutive expression of CD40 in WT mice (lungs, eyes, pancreas, mesentery, and distal colon), expression of CD40 was not detected (data not shown). The absence of tissue activity of 125I-labeled anti-CD40 MAb in CD40−/− mice indicates that the selective accumulation of this antibody can be attributed to specific MAb binding to its ligand.
In a separate group of experiments, we assessed whether CD40L, the natural ligand for CD40, influences the basal expression of CD40 on vascular endothelial cells. This was accomplished by measuring the expression of CD40 in unstimulated CD40L deficient (CD40L−/−) mice. As shown in Fig. 1, the expression of CD40 is either profoundly reduced or absent in the heart, kidneys, brain, and small intestine of CD40L−/− mice. A similar diminution of CD40 expression was also noted in other tissues of CD40L−/− mice.
Cellular sources of CD40L that modulate CD40 expression.
CD40L is found predominantly on platelets and T lymphocytes. Therefore, experiments were performed to address whether platelet- and/or T-lymphocyte-associated CD40L represent the source of CD40L that elicits the basal expression of CD40 on vascular endothelial cells. This possibility was examined by measuring the constitutive expression of CD40 in WT animals rendered thrombocytopenic (APS group) as well as RAG-1−/− mice, which are genetically deficient in lymphocytes. Treatment with APS resulted in a significant reduction (>92%) of platelets in the peripheral blood compared with levels before APS administration. Figure 2 compares the levels of constitutively expressed CD40 in the heart, kidneys, brain, and small intestine of WT controls, APS-treated WT mice, and RAG-1−/− mice. In the absence of platelets (and platelet-associated CD40L), CD40 expression was significantly attenuated in the heart and kidney and was undetectable in the vasculature of the brain and small intestine. Similarly, in animals lacking T lymphocytes (and T-cell-associated CD40L), the heart vasculature exhibited a significant reduction in CD40 expression, whereas the kidney, brain, and small intestine demonstrated a complete absence of this receptor. A decrease in constitutive CD40 expression in mice lacking platelet or T-cell-associated CD40L was also noted in other tissues (data not shown).
LPS-induced expression of CD40.
Figure 3 illustrates the time course and magnitude of CD40 expression in the heart, kidneys, brain, and small intestine after LPS challenge. Significant differences in the time course and magnitude of anti-CD40 MAb accumulation were noted between tissues. An increased CD40 expression was observed in the heart, kidneys, and small intestine at 4 h after LPS challenge, and CD40 expression largely remained at this level for up to 48 h. The kidney exhibited the most robust response with a sevenfold increase over constitutive levels after LPS administration, and approximately four- to fivefold increases were noted in the heart and small intestine. In contrast to other tissues studied, the increase in expression of CD40 in the brain (3.5-fold, Fig. 3C), lung (2.5-fold, data not shown), and the eyes (8-fold, data not shown) was delayed and only observed at 48 h after LPS administration. The stomach failed to exhibit any CD40 expression, even after LPS stimulation.
LPS-induced CD40 expression in CD40- and CD40L-deficient mice.
Figure 4 compares CD40 expression measured in the vasculature of the heart, kidneys, brain, and small intestine in WT, CD40−/−, and CD40L−/− mice at 4 h after intraperitoneal injection of LPS. LPS induced a significant increase in CD40 expression in the heart, kidneys, and small intestine of WT mice after 4 h. As expected, CD40 expression was largely undetectable in the vasculature of CD40−/− mice. In contrast to the results obtained from unstimulated mice (Fig. 1), significant CD40 expression was detected in CD40L−/− mice at 4 h after LPS injection. Indeed, the levels of CD40 expression detected in the heart and kidneys of LPS-challenged CD40L−/− mice were comparable to those seen in WT mice. It is noteworthy that the LPS-elicited induction of CD40 was more pronounced in the brain and small intestine of CD40L−/− mice, when compared with WT mice. A similar exaggerated response to LPS was observed in the distal colon of CD40L−/− mice.
The CD40/CD40L pathway has rapidly emerged as an important modulator of the inflammatory and thrombogenic responses that are associated with different acute and chronic disease processes including stroke, atherosclerosis, psoriasis, and inflammatory bowel diseases (17). Ligation of CD40 on endothelial cells confers a proinflammatory phenotype that is characterized by an increased expression of chemokines, cytokines, and adhesion molecules, all of which promote the recruitment of inflammatory cells to the vascular wall (8, 19, 36). This pivotal immunoregulatory function of endothelial cell CD40 has led to efforts to define its histochemical localization on vascular endothelial cells in different tissues (39) and on monolayers of cultured microvascular endothelial cells (25). Although these studies have revealed that CD40 is basally expressed on endothelial cells in a variety of tissues and that its expression can be increased when cultured endothelial cells are activated with cytokines, there is relatively little quantitative information in the literature concerning the distribution and density of expression of this important endothelial receptor in different tissues, and less is known about the factors that regulate endothelial cell CD40 in vivo. The results of the present study provide the first quantitative in vivo evidence that there are significant differences in the constitutive expression of CD40 on endothelial cells among tissues. We also demonstrate a tissue-specific, time-dependent upregulation of endothelial CD40 in mice challenged with bacterial endotoxin, and our study provides novel insight into the role of platelet- and T-lymphocyte-associated CD40 ligand in regulating the expression of CD40 on endothelial cells.
The dual radiolabeled MAb technique employed in this study of endothelial CD40 expression has been previously used to quantify the expression of different proteins, including adhesion molecules (e.g., ICAM-1 and P-selectin) and receptors (e.g., angiotensin II type-1 receptor), on vascular endothelial cells in various tissues of the rat and mouse (21, 31, 32). This method measures the relative accumulation, in any regional vascular bed, of a binding MAb to a specific endothelial surface epitope (e.g., CD40) and an isotype-matched nonbinding MAb, the latter of which is used to compensate for nonspecific accumulation of the binding MAb. Previous studies employing the dual radiolabeled MAb technique have shown that 1) the radiolabeling procedure does not alter the blocking function of the MAb (31), 2) expression of the targeted endothelial cell surface epitope on circulating cells (e.g., CD40 on B cells) does not interfere with the assay because all residual blood is flushed from the vasculature before tissue sampling, and 3) mice that are genetically deficient in the relevant endothelial cell surface epitope yield expression values that are essentially zero under both basal and stimulated conditions (12). In the present study, we demonstrate that although significant basal and induced expression of CD40 is detected in the vasculature of several tissues in WT mice, CD40-deficient mice do not exhibit significant levels of either constitutive or induced CD40 expression, which supports the validity and resolution of the dual radiolabeled MAb method for detecting CD40 expression in vivo.
Our finding of significant basal expression of CD40 in several vascular beds is consistent with previous immunohistochemical studies (39) that demonstrate the presence of CD40 on the vascular endothelial cells of human lung, kidney, spleen, skin, and skeletal muscle. A comparison of CD40 expression in different tissues to the expression of a noninducible endothelial CAM, such as PECAM-1 (Table 1), allows us to normalize the CD40 data for differences in vascular surface area (11, 21). This analysis revealed that the highest density of CD40 on vascular endothelial cells is found in the lung, small intestine, kidney, brain, and heart, whereas other tissues, like the liver, stomach, and colon, express negligible amounts of the receptor under basal conditions. It is possible that endothelial cells in lungs and small intestine express high basal levels of CD40 because these cells are constantly exposed to environmental bacterial antigens. The importance of CD40 in mediating the responses to inflammation or infection in these organs is highlighted by the fact that CD40−/− mice more readily succumb to lung infection (27) or inflammation of the gut (14) after stimulation with bacterial antigens.
Immunohistochemical staining of endothelial CD40 in different diseased tissues, such as tumors and atherosclerotic lesions, have revealed an increased expression of the signaling molecule (5, 30), as have studies of cultured endothelial cells stimulated with cytokines or LPS (29). However, our study provides the first quantitative in vivo assessment of the magnitude and time course of CD40 upregulation in response to an inflammatory stimulus. We have demonstrated a tissue-specific, time-dependent upregulation of endothelial CD40 in mice challenged with bacterial endotoxin. The kidney exhibited the most robust (sevenfold) increase in CD40 expression, whereas most other vascular beds responded with two- to fivefold increases that are similar in magnitude to the CD40 upregulation previously noted on cultured endothelial cells activated with LPS or cytokines (25, 39). Although an explanation for the differences in time course and magnitude of CD40 expression between tissues is not evident from our data, it likely reflects organ-to-organ differences in the responsiveness of endothelial cells and perivascular auxiliary cells (e.g., mast cells and macrophages) to LPS-induced activation. Indeed, the kinetics of LPS-induced CD40 induction in most tissues is comparable to that previously reported for murine endothelial CAMs, such as ICAM-1 and VCAM-1 (21). This similarity in expression kinetics may reflect the shared involvement of the transcription factor NF-κB in the synthesis of both CD40 and these CAMs (15, 26). In the presence of CD40L, it is plausible that LPS may also promote the engagement of platelet- or lymphocyte-associated CD40L with endothelial cell CD40, which subsequently induces the synthesis of additional CD40 as well as CAMs by endothelial cells. This possibility is addressed below.
A novel and potentially important observation in our study was the absence of constitutive CD40 expression in different vascular beds of mice that are genetically deficient in CD40L. Such a response might be expected if the targeted disruption of the gene for CD40L is also accompanied by the inadvertent ablation of the CD40 gene. However, this seems unlikely because of our finding that CD40L−/− mice exhibited substantial CD40 upregulation on endothelial cells after LPS stimulation. A more likely explanation is that there is cross talk between CD40 and CD40L on endothelial cells. This possibility is supported by recent reports describing an increased expression of CD40L on endothelial cells when either platelet- or monocyte-associated CD40L engages with CD40 on cultured endothelial cells (6, 38). The findings of our study suggest that endothelial expression of CD40 and CD40L are in an autoregulatory loop, such that, in the absence of CD40L engagement with CD40, the surface expression of the latter is reduced. Such a regulatory mechanism is similar to what has been proposed for IL-2 and its receptor, the IL-2Rα chain (9). In IL-2−/− mice, the expression of IL-2Rα is absent on activated CD4+ T cells, suggesting that IL-2 is the most critical cytokine controlling the expression of its receptor IL-2Rα (10). Our own data indicate that CD40L is a major determinant of the basal level of CD40 expression on endothelial cells.
To ascertain the source of the CD40L that is regulating basal CD40 expression on endothelial cells, we focused on the two major cellular sources of CD40L in the circulation, T-lymphocytes and platelets (4, 19). CD40L is also shed from activated platelets and T lymphocytes, generating a soluble form of CD40L (sCD40L) (20). Our results indicate that the depletion of either cellular source of CD40L results in a highly significant downregulation of CD40 expression on the vascular wall in almost every organ analyzed. Although >95% of the circulating CD40L exists on platelets (2, 3) and platelets are also the major source of sCD40L in the circulation (23), it is noteworthy that the degree of attenuation was comparable in animals deficient in either platelets or T lymphocytes. Furthermore, the levels of CD40L on both of these cell populations under control conditions are very low or not detectable. Therefore, the exact mechanism of control of constitutive CD40 by CD40L remains unclear. One possibility is that a critical but low density of CD40L expression or a very small percentage of CD40L-expressing cells is required to sustain the basal level of CD40 on endothelial cells. Removal of either cell population may lower the CD40L concentration below the threshold level required to sustain CD40 expression. Although it remains unclear whether this CD40L is cell-associated or the circulating soluble form, the role of sCD40L as a biologically active ligand for CD40 remains controversial, and in the absence of quantitative detection assays for murine sCD40L, this possibility cannot be addressed experimentally at the present time.
A recent study (23) demonstrated that platelets also constitutively express CD40. Therefore, an alternative explanation for the reduction of CD40 expression in thrombocytopenic mice is that platelet-associated CD40 is responsible for the majority of the CD40 expression that we observed in the control mice. However, these experiments were performed under baseline conditions wherein the firm adhesion of platelets to the vasculature is extremely low. Therefore, CD40 expression on a limited number of platelets that may remain adherent to the endothelium in the control mice after flushing out the blood would be unlikely to account for the relatively high constitutive expression of CD40 shown in Figs. 1 and 2.
In contrast to our findings under baseline conditions, our data indicate that CD40L does not contribute to the LPS-induced increase in CD40 expression observed in different vascular beds because CD40L-deficient animals exhibited a normal or even more intense (vs. WT mice) upregulation of CD40 in response to LPS. This precludes a role for platelet- or T-lymphocyte-associated CD40L in endotoxin-mediated CD40 upregulation. Two possible mechanisms for the CD40L-independent upregulation of CD40 expression during endotoxemia are worthy of consideration. First, TNF-α is a major mediator of LPS-induced inflammation (1, 18), and the cytokine is capable of upregulating CD40 expression in vitro (22). Because CD40L is a member of the TNF gene family and both CD40L and TNF-α share several features of endothelial cell activation (20), it is possible that the TNF-α released in the CD40L−/− mice after LPS challenge mimics the biological response of CD40L on endothelial cells by promoting CD40 upregulation. Second, LPS, per se, has been recently shown to be a strong inducer of CD40 expression on macrophages, microglia (33), and endothelial cells (29). Although not well characterized in endothelial cells, LPS induction of CD40 expression in macrophages and microglial cells occurs in a time-dependent manner at the level of transcription and involves the direct activation of the transcription factor NF-κB and an increased production of interferon-γ, with the latter activating STAT-1α, which ultimately resulted in CD40 gene expression (33). However, these data do not exclude a role for CD40L interactions with CD40 to generate subsequent responses in vivo, such as ingestion of bacteria (35), cytokine production, and adhesion molecule expression (29). Regardless of the mechanisms involved in CD40 upregulation during endotoxemia, this endothelial cell response appears to play an important role in regulating the inflammatory response associated with pathological conditions such as sepsis.
Interestingly, in polymicrobial sepsis, where other bacterial products are present, it has been proposed that these products may bind directly to CD40. In fact. E. coli heat shock protein-70 (HSP-70) was shown to stimulate CD40-dependent cytokine production both in vitro and in vivo if the cells or animals are primed with LPS (28). In the same study, it was shown that cecal ligation and puncture-induced mortality is reduced in mice lacking CD40 but not CD40L. Our study provides further information for a CD40L-independent modulation of CD40 responses by demonstrating CD40L-independent upregulation of endothelial CD40 expression after LPS. To date, the CD40-CD40L pathway has been targeted for inhibition largely through the use of CD40L-blocking antibodies. Our findings suggest, however, that under conditions such as sepsis, CD40L immunoneutralization may incompletely or fail to block the CD40-signaling cascade, which should be considered when designing future clinical trials that are directed toward interference with the CD40-CD40L signaling system.
This study was supported by National Institutes of Health Grants DK-065649 and HL-26441 (to D. N. Granger) and the Deutsche Forschungsgemeinschaft Grant VO998/1-1 (to T. Vowinkel).
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- Copyright © 2006 by the American Physiological Society