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Departments of Pharmacology & Experimental Therapeutics (D.K., N.G.P., M.L., M.H., K.K.-G., B.M., W.B., S.C., T.C.T.), Biochemistry (K.K.-G., T.C.T.), Obstetrics and Gynecology (A.A.), and Internal Medicine (T.C.T.), Tufts University School of Medicine and Tufts-New England Medical Center, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: T. C. Theoharides, Ph.D., M.D., Department of Pharmacology, and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111. E-mail: Theoharis.Theoharides{at}tufts.edu.
Abstract
Stress activates the hypothalamic-pituitary-adrenal axis through CRH, leading to production of glucocorticoids that down-regulate immune responses. However, acute stress also has proinflammatory effects. We previously showed that restraint stress, as well as CRH and its structurally related urocortin (Ucn), could activate mast cells and trigger mast cell-dependent vascular permeability. Here we show for the first time that human cord blood-derived cultured mast cells (hCBMC) at 10 wk, but not at 2 wk, are immunocytochemically positive for CRH and Ucn; human leukemic mast cells are weakly positive for both peptides. The ability of these mast cells to synthesize CRH and Ucn was confirmed by showing mRNA expression with RT-PCR. hCBMC (8–14 wk) synthesize and store 1–10 ng/106 cells (10–20 µg/g) of both CRH and Ucn detected by ELISA of cell homogenates. Stimulation of IgE-sensitized hCBMC with anti-IgE results in secretion of most CRH and Ucn. These findings indicate that mast cells are not only the target, but also a potential source of CRH and Ucn that could have both autocrine and paracrine functions, especially in allergic inflammatory disorders exacerbated by stress.
STRESS ACTIVATES THE hypothalamic-pituitary-adrenal axis through the release of CRH leading to secretion of catecholamines and glucocorticoids; these, in turn, suppress the immune response (1). CRH is a 41-amino-acid peptide synthesized in the hypothalamus and mediates its effects through at least two types of receptors (R): CRHR-1, CRHR-2, of which a number of subtypes have been identified. These receptors are also present in other brain regions and outside the central nervous system including immune cells (1); therefore, CRH, or structurally related compounds such as urocortin (Ucn) (2), could have immunoregulatory actions (3). However, stress also worsens a number of neuroinflammatory disorders through proinflammatory effects of CRH (4) that are apparently mediated by mast cell activation (5). For instance, acute restraint stress induces intracranial rat mast cell activation (6) that is CRH-dependent as it is blocked by the nonpeptide CRHR-1 antagonist Antalarmin (7). Stress also increases blood-brain-barrier permeability (8) that is dependent on mast cells and CRH (9). Moreover, CRH (5) and Ucn (10) induce mast cell degranulation and vascular permeability in rodent skin, actions mimicked by acute restraint stress (11), but absent in W/Wv mast cell-deficient mice and also blocked by Antalarmin (5). Acute restraint stress also increases skin CRH levels (12); it also increases skin mast cells and exacerbates delayed hypersensitivity reactions through CRHR-1 (13).
Mast cells are ubiquitous in the body and are critical for allergic reactions, as they secrete numerous vasoactive molecules and cytokines (14, 15). Increasing evidence, however, indicates that mast cells also secrete proinflammatory cytokines (14, 15), such as IL-6 (16) and are involved in neuroinflammatory processes (17). These findings have prompted the speculation that mast cells may have a much more versatile role than previously suspected (18, 19). It has been difficult to obtain human mast cells, as they are usually isolated from clear margins of lung resections or from foreskin following circumcisions. Most of our current knowledge about mast cell-derived cytokines comes from human leukemic (HMC-1) mast cells, but these cells do not respond to physiologic triggers, such as stimulation by antigen or anti-IgE, because they do not express IgE binding protein (Fc
RI) (20). A number of investigators have developed and used human umbilical cord blood-derived mast cells (hCBMC) grown in the presence of stem cell factor (SCF) and IL-6 (21) for about 16 wk. Sensitized hCBMC respond adequately to anti-IgE stimulation and secrete histamine, tryptase, and a variety of cytokines (21, 22).
Here, we show for the first time that human umbilical cord blood-derived cultured mast cells and HMC-1 cells synthesize both CRH and Ucn, which they can secrete in response to immunologic stimulation.
Materials and Methods
Human mast cell culture
Human umbilical cord blood was obtained from healthy mothers during normal deliveries, with approval by the Hospital’s Human Investigation Review Board and without any identifying information or other medical or social history of the mother. The hCBMC were generated as described previously with minor modifications (21). Briefly, mononuclear cells were isolated by centrifuging heparin-treated cord blood onto lymphocyte separation medium (INC Biomedical, Aurora, OH) at 350 x g for 30 min at room temperature and the buffy coat was removed. Mast cell progenitors were then isolated by positive selection of AC133 expressing cells (CD133+) by an immunomagnetic cell sorting method (Miltenyi Biotec, Auburn, CA). The CD133 is a surface antigen with a molecular mass of 177 kDa, which is selectively expressed on a CD34bright (CD34+) subset of human hemopoietic stem and progenitor cells. The purified CD34+ cells were cultured in Iscove’s modified Dulbecco’s medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 100 ng/ml recombinant human SCF (a generous gift from Amgen, Thousand Oaks, CA), 50 ng/ml IL-6 (Chemicon International, Inc., Temecula, CA), 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 50 µM 2-mercaptoethanol (GIBCO BRL), 1% penicillin-streptomycin (GIBCO BRL) at 37 C in a 5% CO2 incubator. Both SCF and IL-6 are required for normal growth and maturation of hCBMC (21). Cultured hCBMC respond adequately to immunologic stimulation for up to about 16 wk. The hCBMC used in this study were 10 wk old for the positive immunocytochemistry and RNA extraction, but were from 8–14 wk for the ELISA studies because more cells were required. The purity of mast cells reached greater than 99% by May-Gruenwald and Giemsa Staining and were 100% tryptase positive by 8 wk of culture.
HMC-1 cell culture
HMC-1 cells were kindly supplied by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) and were cultured as previously described (23).
Immunocytochemistry for CRH and Ucn
Cytospin smears of cells (hCBMC and HMC-1) were prepared using Cytospin 3 (Shandon, Pittsburgh, PA) and were stained immunohistochemically for CRH and Ucn by peroxidase using the Dako EnVision System, Peroxidase Kit (Dako Corp., Carpinteria, CA) as per the directions of the manufacturer. Cytospin smears were fixed with Carnoy’s solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 3 min. Slides were then incubated with peroxidase blocking reagent (0.03% hydrogen peroxide containing sodium azide) for 5 min and placed in Tris-buffered saline (TBS) for 5 min. The primary antibodies used were polyclonal rabbit antihuman CRH (Phoenix Pharmaceuticals, Inc., Belmont, CA) and polyclonal rabbit antihuman Ucn (Phoenix) at 1:60 and 1:500 dilutions for 30 min. Slides were placed in TBS for 5 min and incubated with peroxidase-labeled polymer conjugated to goat antirabbit and antimouse Igs for 30 min. Slides were then rinsed with TBS and incubated with substrate 3,3'-diamino-benzidine chromogen solution for 5 min. All the incubations were carried out at room temperature. Presence of a brown-colored end product at the site of the target antigens indicates positive reactivity. Negative controls contained samples in which the primary antibody was omitted or replaced by normal rabbit serum, as well as immature hCBMC of 2-wk culture. HMC-1 were also included for comparison.
Total RNA extraction and RT-PCR for CRH and Ucn mRNA expression in mast cells
Total RNA was prepared from 10-wk-old hCBMC and HMC-1 cells using the TRI Reagent (Sigma, St. Louis, MO). Cells (107) were collected by centrifugation and lysed in 1 ml TRI reagent according to the manufacturer’s recommendations for RNA isolation from suspension cells. First-strand cDNA synthesis was performed from 2 µg total RNA (heated for 3 min at 65 C) using 200 U murine Moloney virus reverse transcriptase, 500 µg/ml oligo deoxythymidine primer, 40 U/µl RNaseOUT recombinant ribonuclease inhibitor (Invitrogen Life Technologies, Carlsbad, CA) and 5 mM deoxynucleotide triphosphates (Roche Diagnostics, Mannheim, Germany). The reaction (20 µl) was run for 50 min at 37 C, and inactivated by heating at 72 C for 15 min. A portion of cDNA (10%) was used for amplification in PCR for 30 cycles. Two specific oligonucleotide sets of primers, previously published for CRH (sense: 5'-TTCCGAGGAGCCTCCCATC-3' and antisense: 5'-AATCTCCATGAGTTTCCTGTTGC-3' (24) and Ucn (sense: 5' CAGGCGAGCGGCCGCG-3' and antisense: 5'-CTTGCCCACCGAGTCGAAT-3' (25) mRNA detection, were employed in this study using the same amplification conditions with the following modifications: all PCRs (50 µl volume) were carried out using 1 U Taq DNA Polymerase (Invitrogen Life Technologies), 0.2 mM deoxynucleotide triphosphates, 6 pmol of each primer, 4.5 mM MgCl2 (Invitrogen Life Technologies) for CRH and 3.5 mM MgCl2 for Ucn, in a DNA Engine-Peltier Thermal Cycler (MJ Research, Watertown, MA). Placental and whole uterus cDNAs (CLONTECH, Palo Alto, CA) were used as positive controls for CRH and Ucn PCR amplification respectively; cDNA was replaced with sterile distilled water as negative control for all PCRs. The amplified product was electrophoresed on a 2% agarose gel and visualized by ethidium bromide, using 100-bp DNA ladder (Invitrogen Life Technologies) to estimate the band sizes, which were 122 bp for CRH and 145 bp for Ucn.
Activation of hCBMC with anti-IgE
For anti-IgE stimulation experiments, hCBMC (106 cells/ml) were washed with Iscove’s modified Dulbecco’s medium and Tyrode’s buffer, once in each, and passively sensitized by incubation with human myeloma IgE (2 µg/ml/106 cells; Chemicon Inc.) for 48 h in culture medium at 37 C. Cells were then washed two times and resuspended in fresh culture medium. Cells were stimulated with anti-IgE (Dako) at 15 µg/ml in 96-well round bottom culture plates (2 x 105 cells in 200 µl medium/sample) for 6 h at 37 C in 5% CO2 as we previously reported (22). Tryptase was measured in the supernatants and cell pellets by fluoroenzyme-immunoassay (Pharmacia, Uppsala, Sweden) as reported previously (26). CRH, Ucn, and tryptase results were expressed as pg/ml and as percent of total = [supernatant/(supernatant + pellet)] x 100%. This presentation allowed normalization of any baseline differences between cultures. These peptides were measured in the supernatant and pellet to calculate percentage of release.
Assay for CRH and Ucn by ELISA
CRH and Ucn were measured in the supernatant or the cell pellet after cells were lysed in lysis buffer (0.5% Triton X-100 containing Aprotinin and 100 mM PMSF in PBS) with sonication three times for 15 sec each at full power (Dimco Gray Co., Dayton, OH; model no.161) at 4 C. CRH and Ucn were assayed by ELISA (Phoenix Pharmaceuticals, Belmont, CA).
Statistics
Results are expressed as mean ± SD and similar groups were compared using Student’s t test. Results from different cultures were compared using ANOVA. Significance is denoted by P < 0.05.
Results
Immunocytochemistry for CRH and Ucn in hCBMC and HMC-1 cells
Cultured hCBMC respond adequately to immunologic stimulation from 8–16 wk of culture. For immunocytochemistry, we compared 2 and 10-wk-old cells. hCBMC and HMC-1 cells were immunostained with specific antibodies to CRH (Fig. 1
, A, C, and E) or Ucn (Fig. 1, B, D, and F). The cytoplasm of 10-wk-old hCBMC (Fig. 1, A and B) was uniformly stained brown with antibodies to both CRH and Ucn, indicating that hCBMC contain both peptides. Negative controls did not show any immunoreactivity for either peptide (not shown) and included 2-wk-old hCBMC that were almost negative (Fig. 1, C and D). HMC-1 cells were moderately stained for both peptides (Fig. 1, E and F). hCBMC and HMC-1 cells without primary antibody or by using normal rabbit serum showed no positive staining for either CRH or Ucn (results not shown).
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) and 145 bp for Ucn (Fig. 2B). Positive controls included placental cDNA and whole uterus cDNA for CRH and Ucn, respectively; they were previously shown to express these PCR products using the same sets of primers. No amplification product was detected in the negative control as shown in Fig. 2 (blank) in which cDNA was substituted with water. PCR amplification product using primers for ß-actin as a housekeeping gene did not show any variation in the cDNA content among these samples (results not shown).
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). The corresponding values for Ucn were 1.2 ± 0.6 ng/106 hCBMC, and 0.9 ± 0.3 ng/106 HMC-1 cells, respectively.
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) from a spontaneous level of 63.7 ± 45.3 ng/5 x 105 cells (3.2%) to 360.3 ± 144.1 ng/5 x 105 cells (27.8%) (n = 6, P < 0.05). Similar stimulation induced considerable secretion of CRH (Table 1
) from spontaneous release of 0.06 ± 0.01 ng/106 cells (5.4%) to 0.8 ± 0.2 ng/106 cells (41.9%). Ucn secretion was also increased from spontaneous release of 0.05 ± 0.02 ng/106 cells (4.1%) to 0.8 ± 0.3 ng/106 cells (69.2%, P < 0.05).
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This is the first time that CRH and Ucn are shown to be expressed, synthesized, stored, and secreted from human umbilical cord-blood-derived mast cells. The amount of CRH and Ucn in hCBMC was 1–10 ng/106 cells and varied almost 10-fold among the different primary cultures. The reason for the observed variability on the levels of CRH/Ucn in this study might be in part due to a wide variation among the individual batches of hCBMC, as previously reported for the difference in mediator secretion from hCBMC (27, 28), or the duration of culture of different batches of cells. This variability may be due to genetic factors, as well as the allergic or stress history of the mother/infant that were not available to us during umbilical cord blood collection. Using the average weight of one mast cell as 500 pg (see Table 1), the maximal CRH and Ucn content is calculated to be approximately 10–20 µg/g. This amount of CRH is approximately 1000 times more than what has been reported for human placenta at term (1 ng/g) (29), more than 100 times higher than what was reported for the hypothalamus (106 ng/g) (30), and more than 10 times higher than what has been reported for carcinoid tumors (1000 ng/g) (31) (Table 1). It appears that these cultured mast cells may be one of the richest sources of CRH and Ucn reported to date; however, the recent advances in assay sensitivity makes difficult to make direct comparisons with older reports and such advances may account for the different levels reported. Moreover, our conclusion that mast cells may be one of the richest sources may not necessarily reflect tissue mast cell levels because hCBMC may: 1) lack any inhibitory signals otherwise provided by the tissue microenvironment and 2) have CRH/Ucn up-regulated by the IL-6 required for hCBMC culture. The mast cell content of Ucn is almost 100 times more than that reported in human peripheral lymphocytes (300 pg/108 cells) (32) that, unlike mast cells, produce only Ucn and not CRH (33). These results, along with the previous finding that HMC-1 (Ref. 5 and unpublished observations) and articular mast cells also express CRHR-1 (34), indicate that CRH and Ucn may also have autocrine effects on mast cells.
Mast cells are found everywhere in the body and are necessary for allergic and late phase reactions because they secrete many vasoactive molecules and cytokines (14, 15). Recent evidence indicates that mast cells may also be involved in neuroinflammatory conditions (17) and may play a much more versatile role than previously reported (18). A case in point is rheumatoid arthritis (RA), where mast cells are increased in the joints (35, 36), and express CRH receptors (34). Both CRH and Ucn have been reported to be elevated in the joints of patients with RA (37), a condition known to be exacerbated by stress (38, 39, 40). For instance, CRH was elevated in the synovium of patients with RA (104 ± 33 pg/ml) and was significantly higher than in osteoarthritis (25 ± 4 pg/ml) (41). We recently also showed that neither vascular permeability (42) nor carrageenan-induced arthritis (43) could develop in mast cell-deficient mice. Inflammatory arthritis was also greatly reduced in CRH knockout mice (43), whereas autoimmune arthritis was reduced by Antalarmin (44). Our results are also supported by the recent finding that autoimmune arthritis could not develop in mast cell-deficient mice (45). Mast cells could, therefore, participate in RA both by being the targets of CRH/Ucn secreted from local nerve endings upon stress and by secreting CRH/Ucn themselves. Secretion of CRH/Ucn from mast cells could also be triggered by proinflammatory molecules that are released during the initial phases of inflammation.
Acute restraint-stress led to mast cell-dependent increase in vascular permeability in rodent skin (11), an effect also induced by local administration of CRH (5) and its structurally related Ucn (10); these effects were blocked by the nonpeptide CRHR-1 antagonist Antalarmin. Acute stress also increased the CRH content of skin blister fluid (12), and CRH iontophoresis was recently shown to induce skin vasodilation in humans (46). Moreover, stress-induced alopecia was associated with CRH receptor up-regulation in affected sites (47).
Acute stress also increased the number of skin mast cells and worsened delayed hypersensitivity, effects blocked by pretreatment with a CRH receptor antagonist (13). Acute restraint stress also induced intracranial rat mast cell activation, a CRH-dependent action, as it was blocked by Antalarmin (6) and increased dura vascular permeability that was absent in W/Wv mast cell-deficient mice (48). Moreover, acute stress triggered brain mast cell activation and increased brain vascular permeability (8), an effect dependent on CRH and mast cells as it was absent in mast cell-deficient mice. These findings are relevant in view of the fact that hypothalamic mast cells could stimulate the hypothalamic-pituitary-adrenal axis (49, 50, 51).
In conclusion, the present demonstration that mast cells synthesize and secrete CRH/Ucn reinforces a bidirectional relationship of CRH/Ucn and mast cells in inflammatory conditions (19), especially those exacerbated by stress (52).
Acknowledgments
We thank Amgen (Thousand Oaks, CA) for kindly providing the recombinant human SCF and Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) for the HMC-1 cells. We also thank Ms. Mary Stavropoulos for her patience and word processing skills.
Footnotes
This work was supported in part by NIH Grants NS38326 and AR47652 (to T.C.T.).
Abbreviations: CRHR-1 and -2, CHR receptors 1 and 2; hCBMC, human cord blood-derived cultured mast cells; HMC-1, human leukemic mast cells; RA, rheumatoid arthritis; SCF, stem cell factor; TBS, Tris buffer saline; Ucn, urocortin.
Received June 27, 2003.
Accepted for publication October 15, 2003.
References
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