| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CUTTING EDGE |
* Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
Department of Medicine, University of California, San Francisco, CA 94143;
Department of Rheumatology, Nanfang Hospital, Southern Medical University, Guangzhou, China;
Department of Internal Medicine, Yale University, New Haven, CT 06520;
¶ Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
), smooth muscles cells (SMC), endothelial cells, and mast cells also express this cytokine under inflammatory (3) or antigenic stimulation (4). An involvement of MIF in allergic responses was suggested from observations of increased MIF levels in bronchioalveolar lavage fluid (BALF) from asthmatic patients (5). Eosinophils, the hallmark cells of asthma, release MIF in a time- and concentration-dependent fashion in response to phorbol myristate acetate (5). Further, MIF levels are significantly higher in serum and sputum from asthmatic patients and correlate with the production of eosinophil cationic protein (6), a marker for eosinophilic inflammation of airways in bronchial asthma. It is uncertain, however, whether MIF in asthmatic serum or sputum is a nondiscriminate marker of airway inflammation or directly participates in asthma pathogenesis. In this study, we examined the role of MIF deficiency in a murine model of allergic lung inflammation and demonstrate a pathogenic role for mast cell-derived MIF in airway inflammation and allergy. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
BALB/c MIF-deficient mice C.129S4(B6)-Miftm1Dvd (MIF–/–) were generated by backcrossing MIF–/– mice in C57BL/6J background (7) to BALB/c mice (>12 generations). To induce airway allergic responses, a 12-wk-old mouse was immunized i.p. with 50 µg of OVA in 10 mg of Al(OH)3 on days 0, 7, and 14, followed by intranasal challenge with 1 mg of OVA on days 21, 22, and 23 (8). On day 24, mouse serum collection, BALF harvesting, total leukocyte counting, and Diff-Quik staining (cell typing) were performed as detailed elsewhere (8).
Serum and BALF IgE and cytokine level determination
Serum total IgE was determined with a sandwich ELISA (8). Serum and BALF eotaxin (Cell Sciences), MCP-3 (Cell Sciences), IL-4 (Pierce), IL-5 (Pierce), IL-13 (PeproTech), IFN-
(BioSource International), TGF-
1 (BioSource International), and TNF-
(BioSource International) were determined using ELISA kits according to the manufacturers’ instructions. MIF was measured by a murine-specific, capture ELISA (detection limit of 0.16 ng/ml) (9).
Preparation of lung dendritic cells, splenocytes, CD4+ T cells, and mast cells
Mouse lung dendritic cells (DC) were isolated as described previously (10). DC were verified by FACS analysis for CD11c, DEC-205, I-Ad, CD11b, CD14, F4/80, and Gr-1. Isolated lung DC showed positive staining for the cell surface markers of CD11c, DEC-205, and I-Ad, and negative cell surface staining for CD11b, CD14, F4/80, and GR-1. Splenocytes and CD4+ T cells were isolated from C57BL/6J or DO11.10 mouse spleens as we reported previously (11). Mast cells were derived from bone marrow from both MIF+/+ and MIF–/– mice in the presence of murine IL-3 (PeproTech) as previously reported (12). PWM-stimulated mast cells were prepared by differentiating bone marrow cells in medium containing PWM-stimulated, splenocyte-conditioned medium as described (13).
Mast cell reconstitution
Bone marrow-derived mast cells (BMMC) from MIF+/+ mice were washed with calcium-free PBS and resuspended in PBS. Cells were immediately injected into the tail veins of 5-wk-old MIF–/– mice (1 x 107 cells/mouse). These mice were used in the allergic response model 7 wk after BMMC reconstitution, when BMMC appeared in most recipient mouse lungs (14). Reconstituted BMMC were examined using rabbit anti-murine MIF polyclonal Ab-mediated immunostaining.
Preparation of mouse MIF polyclonal Abs
Full-length mouse MIF cDNA was subcloned into pCRT7/NT-TOPO vector and expressed in BL21 (Invitrogen Life Technologies). Poly(His)6-MIF fusion proteins were purified over a His·Bind Quick column (Novagen), and purified proteins were used for immunizing rabbits to produce polyclonal Abs (Proteintech Group). Ab specificity was verified by M lysate immunoblot analysis and immunostaining of lung paraffin sections from both MIF+/+ and MIF–/– mice.
Lung histology
Mouse lungs were removed and fixed in 10% buffered formalin. Paraffin sections (4 µm) were prepared and used for immunostaining of MIF (rabbit anti-mouse MIF polyclonal Ab; 1/2000) and cell proliferation marker Ki67 (rabbit anti-mouse polyclonal Ab; 1/1000, NovoCastra).
MLR and Ag presentation
MLR was carried as previously described (11) in RPMI 1640 on 96-well plates with 2 x 105 T cells per well from C57BL/6J mice and different amounts APC, including lung DC, alveolar M, resting BMMC, and PWM-stimulated BMMC from BALB/c mice. Ag presentation was performed with 2 x 105 DO11.10 T cells mixed with 2 µg/ml OVA323–339 peptide and different amount of APC. Plates were incubated for 2 days followed by the addition of 1 µCi of [3H]thymidine and another 6 h of incubation before harvesting.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
In accord with what has been found in human asthmatic patients (5), the BALF of OVA-sensitized MIF+/+ mice contained higher amounts of MIF than the saline-sensitized controls (Fig. 1A), whereas the BALF of OVA-sensitized MIF–/– mice contained no MIF (data not shown). Immunohistochemical analysis using an anti-murine MIF polyclonal Ab also revealed increased production of MIF in the airway epithelial cells and in peribronchial inflammatory cells, including mast cells. There was a clearly increased density of peribronchial inflammatory cells in the experimentally induced lungs (Fig. 1B). These initial data suggested a correlation between MIF expression and murine airway inflammation and that a lack of MIF reduces such allergic responses. This hypothesis was examined using MIF–/– mice. In the same model, OVA-sensitized MIF–/– mice showed evidence of reduced total leukocyte infiltration (Fig. 1C), including eosinophils (Fig. 1D) and polymorphonuclear (PMN) cells (Fig. 1E) in the BALF when compared with those in OVA-sensitized MIF+/+ mice. The reductions in M (Fig. 1F) and lymphocytes (Fig. 1G) were not statistically significant (ANOVA t test), suggesting that MIF is not essential for M or lymphocyte recruitment but plays a more dominant role in lymphocyte activation and cytokine production. Eosinophils play a critical role in allergic airway remodeling (15) and often serve as hallmarks of allergic airway inflammation. Reduced eosinophil infiltration in MIF–/– mice thus suggested a direct participation of MIF in airway allergic responses. Along with reduced eosinophil infiltration, serum total IgE levels also were significantly lower in MIF–/– mice after OVA immunization (Fig. 1H). Reduced IgE was not due to altered B cell function, because splenocytes from MIF–/– mice showed similar levels of cell surface I-Ad expression, LPS-induced proliferation, and DO11.10 T cell activation as those from MIF+/+ mice (data not shown). These data in MIF–/– mice unequivocally demonstrated that MIF is not simply a molecule marker of inflammation but rather plays an essential role in the pathogenesis of allergic airway diseases.
|
Reduced inflammatory cell infiltration in MIF–/– mice suggested reductions in chemoattractant production. We anticipated that MIF may affect the production of chemoattractants and indirectly impair leukocyte or eosinophil infiltration into the lung. To test this hypothesis, we measured both eotaxin and MCP-3 in serum and BALF. Eotaxin acts via the CCR3 receptor to mediate the chemotaxis of both eosinophils and mast cells in allergen-sensitized airway inflammation and hyperresponsiveness (16). An eotaxin ELISA demonstrated a significant reduction in this chemokine in BALF, but not in serum, from OVA-sensitized MIF–/– mice as compared with OVA-sensitized MIF+/+ mice (Fig. 2A). MCP-3 is important for eosinophil infiltration and activation and acts via CCR1, CCR2, and CCR3 (17). Consistently, serum but not BALF from OVA-sensitized MIF–/– mice showed a significant reduction of this chemokine (Fig. 2B). Therefore, MIF appears to regulate the production of chemokines to control migration and activation of eosinophils (Fig. 1D) or other leukocytes such as PMN (Fig. 1E).
|
from eosinophils (18) or airway SMC (19). In vitro experiments demonstrated that TGF- induces MIF expression in murine colon carcinoma cells (20) and, in turn, MIF regulates TGF- expression in mesangial cells (21). Therefore, it is possible that reduced numbers of eosinophils in MIF–/– mice may lead to a reduction of BALF TGF- levels, which may explain reduced peribronchial inflammatory cell densities in MIF–/– mice (Fig. 1B) by an impairment in their proliferation. A TGF-1 ELISA affirmed this hypothesis and demonstrated a significant reduction of this multifunctional growth factor in BALF but not in the serum of saline- or OVA-sensitized MIF–/– mice (Fig. 2C). These data suggest a role for MIF in eosinophil and/or airway SMC TGF- production and consequent airway inflammatory cell proliferation. Indeed, the numbers of Ki67-positive cells were reduced in OVA-sensitized MIF–/– lungs relative to OVA-sensitized MIF+/+ lungs (Fig. 2D). MIF deficiency impairs Th1 and Th2 cytokine production
In addition to leukocyte infiltration, T cell activation and cytokine production also play critical roles in the pathogenesis of allergic airway diseases such as asthma. Although asthma is mediated by Th2-type T cells, which produce a repertoire of cytokines including IL-4, IL-5, and IL-13, there is a clear role for Th1 cytokines such as IFN- in both the establishment and direction of the allergic phenotype. Increased MIF levels in human asthmatic serum, sputum, and BALF (5, 6), as well as OVA-sensitized MIF+/+ mouse BALF (Fig. 1A), suggest a role of MIF in maintaining airway inflammation and T cell activities. Therefore, a lack of MIF may lower airway inflammation, as reflected by a reduction of BALF total leukocytes (Fig. 1C) or eosinophils (Fig. 1D) and reduce T cell activity. To test this hypothesis, we measured both the Th1 cytokines IFN- and TNF- and the Th2 cytokines IL-4, IL-5, and IL-13. BALF but not serum IFN- levels were significantly reduced in MIF–/– mice (Fig. 3A), although BALF TNF- was undetectable (data not shown) and serum TNF- levels remained the same (Fig. 3B). Whereas serum levels of IL-4 were undetectable (data not shown) and those of IL-5 were the same between MIF+/+ and MIF–/– mice (Fig. 3D), BALF IL-4 (Fig. 3C), BALF IL-5 (Fig. 3D), and serum and BALF IL-13 (Fig. 3E) were all reduced in MIF–/– mice. Thus, MIF appears to act on both Th1- and Th2-type cells in this model of airway inflammation.
|
Th1- and Th2-type T cells play essential roles in airway allergic responses (22). Increased levels of Th1- and/or Th2-type cytokines in the serum or BALF of subjects with asthma or Ag-sensitized airway inflammation suggest an enhancement of T cell activation. Thus, reduced levels of T cell cytokines in MIF–/– mice (Fig. 3) suggested a suppression of T cell activation presumably due to the lack of MIF. Indeed, MIF is well known to stimulate T cells both in vitro and in vivo (4). T cell activation can be mediated directly by cytokines or via APC such as B cells, DC, M, and mast cells. To test these possibilities, we performed MLR using C57BL/6J CD4+ T cells and Ag presentation using DO11.10 T cells in the presence of OVA323–339 peptide and different APC obtained from MIF–/– mice, including lung DC, alveolar M, and BMMC. Surprisingly, DC from MIF–/– mice activated T cells as well as DC from MIF+/+ mice in both assays (Fig. 4, A and B). In contrast, both M (Fig. 4, C and D) and BMMC (Fig. 4, E and F) from MIF+/+ mice and MIF–/– mice differed in their ability to elicit T cell activation, although cell surface I-Ad levels in DC, M, and BMMC were not affected by MIF deficiency (data not shown). Further, PWM-stimulated MIF–/– BMMC also showed a reduction in T cell activation (Fig. 4G). Impaired T cell activation by MIF–/– BMMC was not due to an alteration in their intrinsic development or function, as we did not detect significant differences in either morphology or IgE-induced degranulation in BMMC from MIF+/+ and MIF–/– mice according to an established protocol (23) (data not shown). These observations suggested a novel function for mast cell and M-derived MIF in T cell activation and potentially airway inflammation. However, MIF from other cells such as eosinophils, airway SMC, airway epithelial cells (Fig. 1B), and even T cells themselves may be equally important to T cell activation and airway allergic responses.
|
Our MLR and Ag presentation assays demonstrated that MIF from M and BMMC, but not from DC, are essential for in vitro CD4+ T cell activation (Fig. 4). Nevertheless, lung DC are important in initiating and maintaining allergic airway inflammation by polarizing naive T cells into either Th1 or Th2 effector cells, and they establish T cell memory and tolerance to inhaled Ags (24). It is possible that DC express insufficient MIF and, therefore, the MIF-related effects are not strongly mediated by these cells. Moreover, it is known that other cells such as eosinophils and airway epithelial and SMC also produce MIF (18, 19) (Fig. 1B) and, therefore, the role of BMMC- or M-derived MIF in T cell activation/proliferation observed from our in vitro pure cell population assay (Fig. 4) may be compensated by MIF released from other cell types in vivo.
Mast cells are important cellular effectors in asthma or acute or chronic airway inflammation, and their role in other inflammatory/autoimmune pathologies is gaining increasing prominence (2). Mice lacking mast cells are resistant to allergen-induced airway inflammation, and the reconstitution of BMMC into mast cell-deficient mice restores the inflammatory phenotype (12, 25). It is thought that mast cells contribute to airway inflammation by enhancing proliferation and cytokine production of multiple T cell subsets via direct mast cell-T cell interactions and by undefined soluble factors (26) such as MIF. Indeed, we detected 52 ± 2.1 ng/ml MIF in the conditioned medium of 1 x 106 resting BMMC, and mast cells in normal lung were also immunoreactive for MIF (data not shown). To examine this hypothesis in vivo, we injected MIF+/+ BMMC into MIF–/– mice and induced airway inflammation. MIF-positive BMMC can be detected within reconstituted and OVA-immunized MIF–/– lung parenchyma (Fig. 5A) and adjacent to the airway (data not shown), but not in nonreconstituted MIF–/– lungs (Fig. 1B, left panel). OVA-sensitized, BMMC-reconstituted MIF–/– lungs contained higher numbers of Ki67-positive cells in the peribronchial tissues than those in nonreconstituted OVA-sensitized MIF–/– lungs (Fig. 5B), providing direct evidence for mast cell-derived MIF in airway inflammatory cell proliferation. Consistent with increased cell proliferation, BMMC reconstitution also restored the airway inflammatory phenotype, including the recruitment of total leukocytes (Fig. 5C), eosinophils (Fig. 5D), and PMN cells (Fig. 5E), although M numbers were not affected (Fig. 5F). Inversely, BMMC reconstitution reduced total lymphocyte numbers in MIF–/– mice (Fig. 5G), similar to what was seen previously (25). Such reduction in BALF lymphocytes did not affect the Ag-sensitized airway immune responses. Indeed, reconstituted MIF–/– mice produced comparable amounts of serum total IgE as MIF+/+ mice (Fig. 5H), suggesting that the lymphocytes in BMMC-reconstituted mice acted comparably to those in MIF+/+ mice. Phenotype recovery in reconstituted mice was not due to excessive mast cells in their lung. Methylene blue staining demonstrated similar numbers of mast cells per lung section in BMMC-reconstituted mice vs wild-type mice (32 ± 5 vs 38 ± 5; p = 0.39, n = 5). Therefore, BMMC-derived MIF is sufficient to initiate T cell activation in this murine allergic response model.
|
are critical to T cell activation and, therefore, M in addition to mast cells may contribute to aluminum-OVA-induced airway inflammation. Further, in contrast to mast cell-deficient mice, mice deficient in the mast cell-activating molecules Igµ (27) and Fc
RI (28) showed impaired eosinophil infiltration relative to their wild-type controls after aluminum-OVA sensitization. Therefore, more complex mechanisms may be involved.
In summary, our study demonstrates that MIF production in the lungs, in part from intrapulmonary mast cells and M, participates directly in allergic airway inflammation by enhancing inflammatory cell recruitment and activating lymphocytes to promote the release of Th1/Th2 cytokines and chemokines.
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was partially supported by a grant from the Sandler Award for Basic Science (to G.-P.S.) and National Heart, Lung, and Blood Institute Grants BI HL60942 (to G.-P.S.), HL075026 (to P.W.F.), and HL48621 (to H.A.C.), and National Institutes of Health Grant AR49610 (to R.B.). 
2 B.W. and X.H. contributed equally to this study.
3 Current address: Department of Medicine, University of California, San Diego, CA 92103.
4 Address correspondence and reprint requests to Dr. Guo-Ping Shi, Cardiovascular Medicine, NRB-7, 77th Avenue Louis Pasteur, Boston, MA 02115. E-mail address: gshi{at}rics.bwh.harvard.edu
5 Abbreviations used in this paper: MIF, migration inhibitory factor; BALF, bronchioalveolar lavage fluid; BMMC, bone marrow-derived mast cell; DC, dendritic cell; M, macrophage; PMN, polymorphonuclear; SMC, smooth muscle cell.
Received for publication October 19, 2005. Accepted for publication August 23, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
|---|
-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice. J. Immunol. 170: 3828-3834. 1 (TGF 1) gene expression by eosinophils in asthmatic airway inflammation. Am. J. Respir. Cell Mol. Biol. 15: 404-409. [Abstract] from airway smooth muscle cells induces autocrine synthesis of collagen. Am. J. Physiol. 280: L999-L1008. 1 expression in experimental IgA nephropathy. Nephrol. Dial. Transplant. 19: 1976-1985.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
