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Departments of
*
Immunology and
Neurology, and
Division of Allergic Disease and Internal Medicine, Department of Internal Medicine, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated the ability of a human IgM mAb designated serum-derived human IgM Ab 12 (sHIgM12) to bind to both human and murine DC in a B7-DC (PD-L2)-dependent manner. This binding resulted in: 1) potentiation of the Ag-presenting ability of the DC, as seen by the ability to activate OT-I TCR transgenic T cells; 2) increase in survival of the treated DC upon cytokine withdrawal; 3) secretion of IL-12; and 4) homing and/or survival of DC, resulting in increased numbers reaching the draining lymph nodes. The loss in ability by a monomeric form of sHIgM12 Ab to potentiate the immune response, together with the finding that the monomer inhibits the activity of intact pentameric Ab, argues that the targeted determinants on the surface of the DC are activated by cross-linking (11, 12).
B7-DC is one of the more recently identified costimulatory molecules belonging to B7 family. B7-DC is expressed on DC and, upon activation, expressed on macrophages (13, 14). PD-1, the receptor for B7-DC (also known as PD-L2), is expressed on T cells upon activation and acts as a negative regulator as a consequence of an ITIM motif present in its cytoplasmic tail (15). However, this receptor-ligand interaction has been demonstrated to result in both inhibition of certain T cell responses (14) as well as activation of others (13). The dual nature of the responses elicited by B7-DC has been attributed to different kinds of stimulation used to modulate T cell function. However, it is also possible that B7-DC binds to additional receptors that mediate alternative functions. This hypothesis has been advanced recently in studies modeling the structure of the B7 family members and binding studies using T cells from PD-1-deficient mice (16, 17).
In this study, we examine the capability of our newly described B7-DC cross-linking Ab (11, 12) to modulate the immune response in vivo using an OVA model of murine allergic airway inflammation. Because cross-linking B7-DC on DC with sHIgM12 Ab resulted in secretion of IL-12 in vitro, a key cytokine that promotes Th1 responses, we reasoned that this Ab treatment might promote a change in the polarizing influence of DC in vivo, resulting in the protection of mice against allergic airway inflammation, and this protective effect was STAT4 dependent. Indeed, systemic administration of Ab before immunization completely protected mice from symptoms and lesions that mimic allergic airway inflammation. Strikingly, this Ab treatment protected mice from allergic symptoms even when administered 14 days after hypersensitization. Moreover, the polarity of T cells isolated from the spleens of therapeutically treated mice was changed from strong Th2 to weak Th1. We conclude that B7-DC cross-linking Ab treatment protects mice in both a prophylactic and, more importantly, a therapeutic setting in a murine model of allergic airway inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old BALBc/J and BALB/c-STAT4tm1Gru mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in the animal house facility, Mayo Clinic, as per the institutional guidelines for further studies. OVA protein was purchased from Sigma-Aldrich (St. Louis, MO). These studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee. The IgM human Ab, sHIgM12, is derived from a patient with Waldenstrom’s macroglobulinemia, as described previously (11). Five hundred milligrams of Ab have been purified. The polyclonal human IgM (pHIgM) Ab used as a control in these studies has been described (18).
Immunization and airway challenge
The sensitization and challenge procedure with OVA was modified from the method described by Zhang et al. (19). Briefly, mice were sensitized by an i.p. injection of 100 µg of OVA adsorbed to 1 mg of alum (Pierce, Rockford, IL). Experimental mice were intranasally challenged with 100 µg of OVA in PBS under tribromoethanol anesthesia.
Treatment with B7-DC cross-linking Ab
In the prophylactic regimen, mice were treated with sHIgM12 or the control pHIgM Ab i.v. at 10 µg per day on days –1, 0, and 1 relative to sensitization with OVA in alum. This schedule was designed to determine whether Ab treatment would prevent the establishment of a Th2-polarized response, typically elicited by the use of alum as an adjuvant. In the therapeutic regimen, the Ab treatments were conducted at the same dose and route on the day before the first intranasal challenge, the day of challenge, and day after challenge with OVA in PBS (days 13, 14, and 15 relative to first sensitization with OVA in alum). This treatment schedule was designed to assess whether treatment with B7-DC cross-linking Ab after immunization with a Th2-polarizing regimen could modulate an established response polarity.
Measurement of airway responsiveness to methacholine
Airway responsiveness was assessed on day 27 by methacholine-induced airflow obstruction in conscious mice in a whole body plethysmograph (Buxco Electronics, Troy, NY). Pulmonary airflow obstruction was measured by enhanced pause (Penh) with a transducer connected to preamplifier modules and analyzed by system software. To measure methacholine responsiveness, mice were exposed for 2 min to PBS, followed by incremental dosages of aerosolized methacholine (Sigma-Aldrich). Penh was monitored for each dose.
Collection of bronchoalveolar lavage (BAL) fluid
Immediately after measuring airway hyperreactivity (AHR), animals were injected i.p. with a lethal dose (250 mg/kg) of pentobarbital (Abbott Laboratories, Abbott Park, IL). The trachea was cannulated, and the lungs were lavaged twice with 0.5 ml of HBSS. After centrifugation, the supernatant was collected and stored at –20°C. The cells were resuspended and counted using a hemocytometer. BAL cell differentials were determined with Wright-Giemsa stain; 
200 cells were differentiated using conventional morphologic criteria. IL-5 in the BAL fluid supernatants was measured by ELISA, as directed by the manufacturer (R&D Systems, Minneapolis, MN).
Histology
After BAL fluid collection, the lung was fixed in 10% formalin and embedded in paraffin. Sections were obtained and stained with H&E, and in some cases with anti-CD3 Ab (using a peroxidase-labeled secondary developing reagent). The sections were evaluated by microscopy at x100 and x400 magnification.
In vitro cytokine production and proliferation
On day 27, splenocytes from the control Ab or the sHIgM12 Ab-treated mice were harvested and processed. Briefly, after making a single cell suspension, RBC were lysed by hypertonic shock using ammonium chloride/potassium bicarbonate/EDTA. Cells were counted and resuspended at 3 million cells/ml in RPMI 1640 (Cambrex, Walkersville, MD). OVA was made to a final concentration of 2 mg/ml and was titrated at half log dilutions. Splenocytes were added at 3 x 105 cells in 100 µl. Supernatants were harvested after 48 h and stored for cytokine assay. Cells were pulsed with [3H]thymidine during the last 18 h of the 72-h assay. Cells were harvested and counted for incorporation of [3H]thymidine (Packard Instrument, Boston, MA).
Stored supernatants were analyzed for IL-4, IL-5, IL-10, IFN-
, and TNF-
by ELISA, per the manufacturer’s protocol (R&D Systems).
Statistical analysis
Data were analyzed using a two-way repeated measures ANOVA or Student’s t test for normally distributed data and the Whitney rank sum test for nonparametric data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treating bone marrow-derived murine DC in vitro with sHIgM12 B7-DC cross-linking Ab resulted in secretion of IL-12 (12). IL-12 is a key cytokine in determining the polarity of the immune response, supporting the Th1 phenotype by promoting the secretion of IFN- (20). Skewing the T cell response from a Th2 toward Th1 phenotype can confer protection in allergic disease such as asthma (21). To test whether cross-linking of B7-DC on murine DC with sHIgM12 Ab results in skewing the immune response away from the pathogenic Th2 phenotype induced by immunization with the adjuvant alum, we analyzed the effect of sHIgM12 Ab treatment during the initial immunization with OVA in the induced asthma-like condition in BALB/c mice. Mice were administered sHIgM12 Ab 1 day before, the day of, and 1 day after immunization with OVA in alum (Fig. 1A). This regimen of Ab treatment results in mild reduction in airway responsiveness to methacholine challenge in comparison with the control Ab-treated mice. Mice treated with the sHIgM12 B7-DC cross-linking Ab, however, were significantly protected from airway responsiveness to methacholine challenge relative to animals treated with isotype control Abs (e.g., Fig. 2A, p = 0.041). The OVA model of allergic asthma is characterized by pulmonary inflammation reflected by a statistically significant increase in the number of total cells in the BAL, an increase in the number of eosinophils in the BAL, and perivascular and peribronchial cellular infiltrates in lung tissue sections (22). The number of total cells in the BAL fluid was significantly reduced in sHIgM12-treated mice (Fig. 2B, p = 0.013). Moreover, sHIgM12 treatment also resulted in prevention of eosinophils migrating to the lungs (Fig. 2C, p = 0.015). This failure to detect significant eosinophilic infiltrates correlated with the reduced levels of IL-5 found following treatment with sHIgM12 Ab (Fig. 2D, p = 0.008). IL-5 is a cytokine that plays a pivotal role in migration of eosinophils (23). Most striking was the finding that sHIgM12 treatment totally abrogated lung inflammation, the thickening of bronchial epithelium, and the accompanying accumulation of mucus plugs that were readily evident in mice treated with isotype control Abs (Fig. 3, A and B).
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Next, we asked whether sHIgM12 Ab treatment could prevent mice from developing allergic airway inflammatory disease in a therapeutic model. In this model, the mice were treated with sHIgM12 Ab on days 13, 14, and 15 following initial sensitization with two priming doses of OVA in alum adjuvant (Fig. 1B). Inflammatory lung disease induced with this regimen was more severe than that induced with a single priming dose of OVA in alum adjuvant. This regimen of Ab treatment provided an opportunity to assess the potential of sHIgM12 Ab to modulate established T cell immune reactivity, in a setting in which the immune response was already skewed toward a pathogenic Th2 polarity. Mice that received the sHIgM12 Ab showed a significant reduction in airway responsiveness to methacholine (Fig. 4A, p = 0.01) relative to animals that received isotype control Ab. The responses of sHIgM12-treated animals to methacholine were comparable to the responses of untreated, naive animals (Fig. 4A). Moreover, the number of cellular infiltrates in the BAL derived from sHIgM12-treated mice was markedly lower than the infiltrates found in BAL from either the control Ab or the PBS-treated mice (Fig. 4B, p = 0.008). The number of cells recovered in the BAL of sHIgM12-treated mice was comparable to the number recovered in normal mice. Similar to our findings with the prophylactic treatment regimen, there was no detectable eosinophilic infiltration in the mice treated therapeutically with sHIgM12 Ab (Fig. 4C, p = 0.001).
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, and the Th2 cytokine, IL-4. The mice that received sHIgM12 showed significantly reduced amounts of both IL-4 (Fig. 4D, p = 0.008) and IFN- (Fig. 4E, p = 0.016) in comparison with the control Ab-treated mice. Although IL-5 levels were not measured in this experiment, the complete absence of eosinophilia suggests that IL-5 levels were most likely low in the sHIgM12-treated animals. This cytokine pattern suggests that sHIgM12 treatment does not lead to a proinflammatory cytokine environment in the lungs by switching the polarity of the T cell response from Th2 to Th1, but rather blocks the development of either kind of T cell response. Immunohistochemistry analysis of lung tissue using CD3-specific Ab as a probe supported this conclusion. Few T cells were present in the lungs of naive animals (Fig. 5A). In contrast, lungs from mice treated with PBS or polyclonal IgM control Ab contained extensive T cell infiltrates (Fig. 5, B and C). Remarkably, the lungs from sHIgM12-treated mice showed no signs of infiltration (Fig. 5D), resembling the lungs of untreated mice (Fig. 5A). In addition, there was no lung pathology in animals that received the therapeutic treatment protocol of B7-DC cross-linking sHIgM12 Ab 14 days after presensitization; the lungs of naive animals (Fig. 3C) and sHIgM12 Ab-treated mice (Fig. 3F) were indistinguishable. In contrast, animals that were treated with PBS (Fig. 3D) or isotype control Ab (Fig. 3E) exhibited severe distortion of their bronchial airways and substantial inflammatory infiltration.
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Treatment with sHIgM12 alters cytokine production in the spleens of allergen-presensitized mice
In the absence of inflammation in the lungs of sHIgM12-treated animals, we examined splenocytes from Ab-treated animals in vitro for the nature of their recall response to OVA challenge to determine whether the Th2 polarity characteristic of an allergic response was altered toward a Th1 polarity. Mice were treated with sHIgM12 Ab or isotype control Ab on days 13, 14, and 15 postsensitization with OVA in alum adjuvant. The splenocytes were harvested at day 28 and were restimulated in vitro with OVA. The proliferative response of T cells in response to Ag was enhanced 10-fold in mice that had received sHIgM12 treatment in comparison with the control Ab treatment (Fig. 6A). This finding is consistent with our previous observation that treatment of DC with our B7-DC cross-linking Ab enhances their ability to stimulate T cells (11). Furthermore, it demonstrates the potential of this Ab to stimulate cellular responses against isolated proteins, an observation that may have important implications for the development of vaccines. The supernatants from the stimulated cultures were harvested and tested for the presence of cytokines. Although mice that received sHIgM12 produced significantly higher levels of IFN- than mice treated with isotype control Ab, neither treatment group produced substantial levels (Fig. 6B, p = 0.008). The same trend was observed for the amount of TNF- produced, in which splenocytes from mice treated with sHIgM12 secrete small amounts of TNF-, while no TNF- was detected in control Ab-treated splenocytes (Fig. 6C, p = 0.029). In contrast to these Th1 cytokines, the prototypic Th2 cytokines IL-4 and IL-5 were substantially lower in cultures from mice treated with sHIgM12 Ab. Splenic cultures from mice treated with sHIgM12 contained very small quantities of IL-4 (Fig. 6D, p = 0.004) or IL-5 (Fig. 6E, p = 0.048). These data indicate that sHIgM12 skews the T cell response toward a Th1 polarity, but that even the Th1 response remains weak, despite a strong proliferative response to secondary Ag challenge. The presence of substantial levels of IL-10 in secondary cultures pretreated with the B7-DC cross-linking Ab sHIgM12 (Fig. 6F, p = 0.008) might explain the absence of inflammation in the lungs of mice challenged intranasally with experimental allergen. Despite the ability of T cells in these animals to secrete IFN- and TNF-, T regulatory cells might dampen this tendency and prevent them from tracking to the lungs. Taken together, these data support the notion that sHIgM12 treatment protects presensitized individuals from allergic airway inflammatory disease by the ability of the Ab to prevent a Th2 type of environment and also due to its ability to promote the secretion of the anti-inflammatory cytokine, IL-10.
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Discussion |
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The mechanism of DC activation by sHIgM12 Ab is not fully understood. We have shown that Ab binding to DC is dependent on expression of the costimulatory molecule B7-DC, and that B7-DC-binding ligands and B7-DC-specific IgG Abs can partially block the binding of the human mAb. Because monomers of the pentameric IgM Ab fail to activate, and in fact block, the activation by the native Ab, we have concluded that the cross-linking ability of the Ab is a critical feature of this reagent. We are now acquiring data (not described in this work) demonstrating conclusively that treatment of cultured DCs (both human and mouse) with sHIgM12 Ab activates specific signaling pathways and reproducibly up-regulates subsets of genes. These new findings tend to support the hypothesis that the systemic immunologic consequences of treatment with sHIgM12 are related to Ab-induced changes in resident DCs. Another, nonexclusive possibility is that Ab administered in vivo interferes with the interaction of B7-DC with a natural ligand. One such ligand is PD-1, a known negative regulator of T cell function (29). The possibility that additional undefined ligands exist is also plausible. Just how disruption of interactions between B7-DC and its ligands in vivo might influence the course of immunity is not known at present.
One possible explanation of the therapeutic effect of sHIgM12 is that B7-DC cross-linking by sHIgM12 Ab might lead to development of new sets of T cells that are skewed to secrete IFN- in response to IL-12 and IFN- secreted by the DC. This new lineage of T cells might then suppress the development of effector Th2 cells from the memory Th2 cell population, and, hence, IL-4 and IL-5 secretion in the vicinity of the lungs (30). The finding that STAT4-deficient mice are not responsive to sHIgM12 Ab treatment is consistent with this hypothesis. However, the possibility that STAT4 is an important signaling intermediary for a B7-DC ligand also remains open to question.
A recent report documents the ability of bone marrow-derived DC to secrete IFN- in response to IL-12 and skew the T cell response toward a Th1 polarity (31). However, we have only detected very small increases in IFN- production in these mice. Spleen cells from Ab-treated mice display a profoundly altered cytokine secretion pattern, suggesting that systemic Ab treatment alters the outcome of subsequent intranasal Ag challenge. Remarkably, no inflammation in the lungs occurs. The up-regulation of IL-10 production by splenocytes suggests the possibility that systemic treatment with sHIgM12 Ab may induce immunomodulatory regulatory T cells that suppress the effector responses in the lungs. A recent report demonstrates the ability of DCs to stimulate T regulatory cells in a costimulation-dependent fashion (32). The details of this kind of response still need to be established.
The possible involvement of PD-1, an inhibitory receptor for B7-DC (PD-L2) expressed by activated T cells, in immunomodulatory effects of sHIgM12 Ab treatment is not known. It remains possible that administration of the IgM Ab blocks interactions between DC and T cells, altering the course of T cell activation. However, it should be noted that very small quantities of Ab are administered during this treatment regimen and the affinity of the Ab for B7-DC is low. In previous experiments, DC treated in vitro with sHIgM12 Ab, and washed, retained the biological effects of treatment even when no Ab was detectable by flow cytometry on their cell surfaces (11). Therefore, we favor the hypothesis that the Ab acts by altering DC function, rather than by blocking interactions between DC and T cells. The view that the Ab acts directly on DC is supported by the finding that sHIgM12 Ab alters the cytokine secretion profile of DC in vitro (12) (S. Radhakrishnan and L. R. Pease, unpublished observations). In a very recent study, i.p. administration of an IgG Ab, specific for B7-DC and F(ab')2 of that Ab, exacerbates airway inflammatory disease, presumably by blocking B7-DC interactions with PD-1 (33). This finding highlights the unique therapeutic value of our systemic treatment protocol using an IgM B7-DC cross-linking Ab.
An important feature of this model is the human origin of the sHIgM12 Ab. Importantly, the Ab binds to human DCs as well as to mouse DCs, and induces comparable intracellular molecular changes in both species in vitro (S. Radhakrishnan and L. R. Pease, unpublished observations). Although it remains to be seen whether systemic treatment of human patients with this Ab can alter the course of asthmatic responses, this is an exciting possibility.
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Footnotes |
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2 S.R. and K.I. made equal contributions to this study.
3 Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Mayo Clinic College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address: pease.larry{at}mayo.edu
4 Abbreviations used in this paper: DC, dendritic cell; AHR, airway hyperreactivity; BAL, bronchoalveolar lavage; Penh, enhanced pause; pHIgM, polyclonal human IgM; sHIgM12, serum-derived human IgM Ab 12.
Received for publication March 2, 2004. Accepted for publication May 7, 2004.
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and antigen-specific T cell activation by dendritic cells. Proc. Natl. Acad. Sci. USA 100:7749.-dependent mechanism. J. Immunol. 172:2530.This article has been cited by other articles:
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) or the B7-DC cross-linking Ab sHIgM12 (•) were challenged with increasing dosages of methacholine. AHR was measured by Penh, as described in Materials and Methods. Data are represented as means ± SEM (n = 10 per group). B, Cellular infiltration in lungs. Mice were sacrificed after measuring the AHR to methacholine. Cells present in BAL were counted. Data are represented as means ± SEM (n = 10/group). C, Eosinophilic infiltration in the lungs. The cells from the BAL fluid were stained with Wright-Giemsa for differential counts. Data are represented as the percentage of eosinophils with respect to the total number of cells counted. D, IL-5 in the BAL. The amount of IL-5 in the BAL was determined by ELISA. Data are represented as means ± SEM (n = 10/group).
) were subjected to airway hyperresponsiveness measurement in response to increasing dosage of methacholine, as in Fig. 1
and biotin-conjugated goat anti-rabbit/avidin-HRP secondary reagents. Sections were analyzed at x400. A, Represents lungs from naive mice. B, Represents lungs from PBS-treated mice. C, Represents lungs from control polyclonal IgM Ab-treated mice. D, Represents lungs from sHIgM12 B7-DC cross-linking Ab-treated mice. Arrows indicate sites of T cell inflammation marked by staining with anti-CD3 Abs.