PUBLIC AGENCIES OPPOSED TO SOCIAL SECURITY ENTRAPMENT et al.
In 1950, Congress amended the Social Security Act to authorize voluntary participation by States in the Social Security System with respect to old age, disability, and death benefits. Under 42 U.S.C. § 418(a) (1982 ed. and Supp.), States may obtain coverage for employees of the State and its political subdivisions by executing an agreement (§ 418 Agreement) with the Secretary of Health and Human Services (Secretary) that is required to be "not inconsistent with the provisions of" § 418. As originally enacted, § 418(g) permitted States to terminate their § 418 Agreements upon giving at least two years' advance notice in writing to the Secretary. However, because the increasing rate of state withdrawals was threatening the integrity of the System, Congress amended § 418(g) in 1983 to provide that no § 418 Agreement "may be terminated, either in its entirety or with respect to any coverage group, on or after April 20, 1983." The amendment expressly prevents States from withdrawing employees from the System even if a termination notice had been filed prior to the amendment's enactment. In 1951, California and the Secretary entered into a § 418 Agreement that covered employees of the State and its political subdivisions. The Agreement recited that its provisions were "in conformity with" § 418, and included a termination clause mirroring the provisions of § 418(g) then in effect. When the 1983 amendment of § 418(g) prevented termination notices that California previously had filed from taking effect, proceedings were instituted in the Federal District Court attacking the validity of amended § 418(g). The court held that § 418(g) was unconstitutional, reasoning that the § 418 Agreement created a "contractual right" in favor of the State and its subdivisions to withdraw from the Social Security System, and that such right constituted "private property" within the meaning of the Just Compensation Clause of the Fifth Amendment. Although the court concluded that amended § 418(g) effected a taking of that property without providing the requisite just compensation, it held that a damages award would be contrary to Congress' will and accordingly simply declared § 418(g) unconstitutional.
Held: Amended § 418(g) does not effect a taking of property within the meaning of the Fifth Amendment. Pp. 51-56.
(a) In enacting the Social Security Act in 1935, Congress anticipated the need to respond to changing conditions, and therefore included § 1304, which expressly reserves to it "[t]he right to alter, amend, or repeal any provision" of the Act. The Act itself, including the original version of § 418(g), created no contractual rights, and therefore Congress had the power to amend that section. In view of the Act's purpose and structure, and of Congress' express reservation of authority to alter its provisions, courts should be extremely reluctant to construe § 418 Agreements in a manner that forecloses Congress' exercise of that authority. Pp. 51-53.
(b) The conclusion that Congress reserved the authority to amend not only § 418 but also § 418 Agreements entered into "in conformity with" § 418 is supported by precedent. Cf. Sinking-Fund Cases, 99 U.S. (9 Otto) 700, 25 L.Ed. 496; National Railroad Passenger Corp. v. Atchison, T. & S.F.R. Co., 470 U.S. 451, 105 S.Ct. 1441, 84 L.Ed.2d 432. The language of § 1304's reservation expressly notified California that Congress retained the power to amend the law under which the Agreement was executed and by amending that law to alter the Agreement itself. Pp. 53-54.
(c) The "contractual right" at issue in this case bears little, if any, resemblance to rights held to constitute "property" within the meaning of the Fifth Amendment. The termination provision in the § 418 Agreement exactly tracked the language of the statute, conferring no right on California beyond that contained in § 418 itself. The termination provision in California's § 418 Agreement did not rise to the level of "property," and thus amended § 418 did not effect a taking within the meaning of the Fifth Amendment. Pp. 54-56.
613 F.Supp. 558 (E.D.Cal.1985), reversed and remanded.
Richard K. Willard, Washington, D.C., for appellants.
Andrew D. Hurwitz, Phoenix, Ariz., for appellees.
On this appeal we review a decision of the District Court for the Eastern District of California that § 103 of the Social Security Amendments Act of 1983, 97 Stat. 71, 42 U.S.C. § 418(g) (1982 ed., Supp. II), effected a taking of property within the meaning of the Fifth Amendment by preventing States from withdrawing state and local government employees from the Social Security System.
As of 1983, more than 90% of the Nation's paid employees, a total of more than 115 million people, participated in the Social Security System. H.R.Rep. No. 98-25, at 13.3 Participation in the System is, and has been since its inception, "basically mandatory." Id., at 19. Therefore, most workers covered by the System and their employers have no choice whether or not to participate. In 1935, when the Act was adopted, Congress faced questions as to whether it could compel the States and their political subdivisions to include their employees in the System.4 Therefore, the Act at that time excluded such employees from its coverage. See 42 U.S.C. § 410(a)(7). Responding to subsequent pressure from States that sought Social Security coverage for their employees, in 1950 Congress enacted § 418, the provision at the heart of the controversy in this case.
Section 418 authorizes voluntary participation by States in the Social Security System.5 Under § 418(a), States may obtain coverage for their employees and employees of their political subdivisions, enrolling all or only specified "coverage groups" of workers. 42 U.S.C. § 418(a)(1) (1982 ed., Supp. II); see § 418(b)(5) (defining coverage group).6 States enter the System by executing "an agreement" (§ 418 Agreement) with the Secretary of Health and Human Services (Secretary).7 While § 418 gives States some authority over the content of the Agreements, i.e., States may identify the covered employees, the provisions of a § 418 Agreement are required to be "not inconsistent with the provisions of" § 418. § 418(a)(1). From its enactment in 1950 through 1983, § 418 permitted States to terminate their § 418 Agreements "[u]pon giving at least two years' advance notice in writing to the [Secretary]." § 418(g)(1). Once a State exercised its option to withdraw, it could not thereafter reenter the System. § 418(g)(3).
Following adoption of § 418, all 50 States entered into § 418 Agreements with respect to their own employees, local government employees, or both.8 "By the early 1960's most States had made coverage agreements," H.R.Rep. No. 98-25, at 18, U.S.Code Cong. & Admin.News 1983, p. 235 and the percentage of state and local employees enrolled in the System increased from 11% in 1951 to 70% in 1970, H.R.Comm. Print 97-34, at 25. Since 1970, "[c]overage of State and local employees has remained fairly constant at 70-72 percent." H.R.Rep. No. 98-25, at 18, U.S.Code Cong. & Admin.News 1983, p. 236. As of 1983, "some 9.4 million out of the approximately 13.2 million State and local employees" participated in the Social Security System. Id., at 17, U.S.Code Cong. & Admin.News 1983, p. 235.
For the first 20 years of their participation, "very few" States exercised their option under § 418(g) to withdraw from the System. Id., at 18. Until the mid-1970's, the number of state and local employees "leaving the system was always greatly exceeded by the number of newly-covered employees—in most years, by 50,000 or more." Ibid.9 Starting in 1976, however, this trend reversed, and the "numbers of positions being terminated from coverage" began to exceed "the numbers of newly-covered positions." Ibid. From 1977 through 1981, "termination activity was greater than in the previous ten years," with coverage "terminated for 96,000 State and local government employees." Ibid. As of 1982, coverage was "terminated for 595 State entities employing 190,000 workers." Ibid. Finally, "for the two-year period of 1983-84, terminations [were] pending for 634 State and local entities employing 227,000 workers." Ibid.
When Congress amended § 418(g) in 1983, California had filed termination notices on behalf of 71 of its political subdivisions, employing approximately 34,000 persons.14 When the amendment prevented the termination notices from taking effect, appellees commenced the lawsuits underlying this appeal, naming as defendants the United States and the Secretary and Undersecretary of the Department of Health and Human Services. The first lawsuit was brought by several public agencies of California, their employees and taxpayers, and by an organization calling itself Public Agencies Opposed to Social Security Entrapment. These parties alleged, among other claims, that amended § 418(g) had deprived them of their "contract rights" without just compensation in violation of the Fifth Amendment.15 In the second lawsuit, the State of California sought to enjoin enforcement of § 418(g) as well as a declaration that the section was unconstitutional. The State claimed that the federal defendants had acted in excess of their constitutional authority and had violated the Tenth Amendment by breaching their contract with the State and by impairing the State's "ability . . . to structure its relationships with its employees."16 App. 26-27.
Ruling on cross-motions for summary judgment, the District Court held that § 418(g) was unconstitutional. Public Agencies Opposed to Social Security Entrapment v. Heckler, 613 F.Supp. 558 (ED Cal.1985).17 The court decided that the § 418 Agreement created a "contractual right" to withdraw from the Social Security System that ran in favor of both the State and its public agencies. This contractual right existed independently of the statutory termination provision, and Congress derived no authority from § 130418 to amend the § 418 Agreement, as opposed to § 418.
The contractual right to withdraw, reasoned the District Court, constituted "private property" within the meaning of the Just Compensation Clause of the Fifth Amendment. Amended § 418(g) effected a taking of that property without providing the requisite just compensation. In the court's view, the "only rational compensation would be reimbursement by the United States to the State or public agencies, of the amount of money they currently pay to the United States for their participation" in the Social Security Program. 613 F.Supp., at 575. Since amended § 418(g) was enacted to solve the Social Security "financial crisis," however, the District Court concluded that an order awarding this measure of damages would be "simply and clearly contrary to the will of Congress." Ibid. Accordingly, the District Court simply declared § 418(g) unconstitutional. Ibid. We noted probable jurisdiction, 474 U.S. 1004, 106 S.Ct. 521, 88 L.Ed.2d 454 (1985), and now reverse.
Congress' decision that American workers need a federal program of social insurance protecting them in old age and disability "has of necessity called forth a highly complicated and interrelated statutory structure." Flemming v. Nestor, 363 U.S. 603, 610, 80 S.Ct. 1367, 1372, 4 L.Ed.2d 1435 (1960). Since the Act was designed to protect future, as well as present, generations of workers, it was inevitable that amendment of its provisions would be necessary in response to evolving social and economic conditions unforeseeable in 1935. Ibid. Congress anticipated that it would be necessary to respond to "ever-changing conditions" with "flexibility and boldness," ibid., and therefore included in the Act "a clause expressly reserving to it '[t]he right to alter, amend, or repeal any provision' of the Act. § 1104, 49 Stat. 648, 42 U.S.C. § 1304. That provision makes express what is implicit in the institutional needs of the program." Id., at 611, 80 S.Ct., at 1373. As appellees must concede, the Act itself, including the original version of § 418(g), created no contractual rights. Cf. Flemming v. Nestor, supra, at 608-611, 80 S.Ct., at 1371-73; see also National Railroad Passenger Corporation v. Atchison, T. & S.F.R. Co., 470 U.S. 451, 465-470, 105 S.Ct. 1441, 1451-1454, 84 L.Ed.2d 432 (1985). Therefore, there is no doubt that Congress had the power to amend the section.
In view of the purpose and structure of the Act, and of Congress' express reservation of authority to alter its provisions, courts should be extremely reluctant to construe § 418 Agreements in a manner that forecloses Congress' exercise of that authority. While the Federal Government, as sovereign, has the power to enter contracts that confer vested rights, and the concomitant duty to honor those rights, see Perry v. United States, 294 U.S. 330, 350-354, 55 S.Ct. 432, 434-36, 79 L.Ed. 912 (1935); Lynch v. United States, 292 U.S. 571, 54 S.Ct. 840, 78 L.Ed. 1434 (1934), we have declined in the context of commercial contracts to find that a "sovereign forever waives the right to exercise one of its sovereign powers unless it expressly reserves the right to exercise that power in" the contract. Merrion v. Jicarilla Apache Tribe, 455 U.S. 130, 148, 102 S.Ct. 894, 907, 71 L.Ed.2d 21 (1982). Rather, we have emphasized that "[w]ithout regard to its source, sovereign power, even when unexercised, is an enduring presence that governs all contracts subject to the sovereign's jurisdiction, and will remain intact unless surrendered in unmistakable terms." Ibid. Therefore, contractual arrangements, including those to which a sovereign itself is party, "remain subject to subsequent legislation" by the sovereign. Id., at 147, 102 S.Ct., at 907.
These principles form the backdrop against which we must consider the District Court's decision effectively to forbid Congress to amend a provision of the Social Security Act. That decision heeded none of this Court's often-repeated admonitions that contracts should be construed, if possible, to avoid foreclosing exercise of sovereign authority. Those admonitions take on added force when the arrangement pursuant to which the Government is claimed to have surrendered a sovereign power is one that serves to implement a comprehensive social welfare program affecting millions of individuals throughout our Nation.
Venerable precedent supports our conclusion that Congress reserved the authority to amend not only § 418 but also Agreements entered into "in conformity with" that section. Just last Term, we considered a statute in which Congress had " 'expressly reserved' its right to 'repeal, alter, or amend' the Act at any time," National Railroad Passenger Corporation, supra, at 456, 105 S.Ct., at 1453, and we noted that the "effect of these few simple words" has been settled since the Sinking-Fund Cases, 99 U.S. (9 Otto) 700, 25 L.Ed. 496 (1879). 470 U.S., at 467-468, n. 22, 105 S.Ct., at 1452, n. 22. The Sinking-Fund Cases involved federal statutes that governed railroads' obligations to the United States on subsidy bonds. The statutes in question expressly reserved Congress' authority to repeal, alter, or amend them, and Congress exercised that power by requiring the railroads to set aside part of their current income as a sinking fund to meet their debts to the Government as those debts came due. The railroads claimed that this amendment deprived them of property without due process and improperly interfered with their vested rights. 99 U.S., at 719, 25 L.Ed. 496. In rejecting those arguments, the Court explained that through the language of reservation, "Congress not only retains, but has given special notice of its intention to retain, full and complete power to make such alterations and amendments as come within the just scope of legislative power." Id., at 720, 25 L.Ed. 496. The effect of the Court's construction of the reservation was to authorize Congress not only to amend the statute granting the railroads' corporate charter but also to change the stipulations of a contract made under that charter subsequently to and independently of the original statute. Whatever the limits of the reserved power, it was "safe to say" that Congress had the authority to provide by amendment whatever rules it might "have prescribed in the original charter" and terms governing the "performance of contracts already entered into." Id., at 721, 25 L.Ed. 496.
This reasoning disposes of appellees' contention that Congress lacked authority to amend California's § 418 Agreement. The State accepted the Agreement under an Act that contained the language of reservation. That language expressly notified the State that Congress retained the power to amend the law under which the Agreement was executed and by amending that law to alter the Agreement itself.19 We have no doubt that in 1950 Congress could have provided that States electing to enter the Social Security System would not have authority to terminate their participation. Therefore, amended § 418(g) falls well within the limits of Congress' reserved power to alter the law governing performance of § 418 Agreements.
The § 418 Agreement provided that its terms were "in conformity with" § 418. Therefore, the Agreement expressly incorporated § 418, which of course was fully subject to Congress' reserved power of amendment. Appellees nonetheless insist that the termination provision embodied in the Agreement constituted a valuable property right that was "taken" when Congress enacted amended § 418(g). In the Sinking-Fund Cases, the Court did observe that Congress' exercise of the reserved power "has a limit" in that Congress could not rely on that power to "take away property already acquired under the operation of the charter, or to deprive the corporation of the fruits actually reduced to possession of contracts lawfully made." 99 U.S., at 720, 25 L.Ed. 496. Similarly, other decisions have held that Congress does not have the power to repudiate its own debts, which constitute "property" to the lender, simply in order to save money. Perry v. United States, 294 U.S., at 350-351, 55 S.Ct., at 434-35; see Lynch v. United States, 292 U.S., at 576-577, 54 S.Ct., at 842.
But the "contractual right" at issue in this case bears little, if any, resemblance to rights held to constitute "property" within the meaning of the Fifth Amendment. The termination provision in the Agreement exactly tracked the language of the statute, conferring no right on the State beyond that contained in § 418 itself. The provision constituted neither a debt of the United States, see Perry v. United States, supra, nor an obligation of the United States to provide benefits under a contract for which the obligee paid a monetary premium, see Lynch v. United States, supra. The termination clause was not unique to this Agreement; nor was it a term over which the State had any bargaining power or for which the State provided independent consideration. Rather, the provision simply was part of a regulatory program over which Congress retained authority to amend in the exercise of its power to provide for the general welfare. Under these circumstances, we conclude that the termination provision in California's § 418 Agreement did not rise to the level of "property." The provision simply cannot be viewed as conferring any sort of "vested right" in the face of precedent concerning the effect of Congress' reserved power on agreements entered into under a statute containing the language of reservation. Since appellees had no property right in the termination clause, amended § 418 did not effect a taking within the meaning of the Fifth Amendment.
The judgment of the District Court is reversed, and the case is remanded for further proceedings consistent with this decision.
According to the Senate Special Committee on Aging, the Social Security System is "much more" than a "retirement program for older workers. . . . Social security is also family security, protecting workers and their families from loss of earnings because of death, retirement, or disability." Senate Special Committee on Aging, Termination of Social Security Coverage: The Impact on State and Local Government Employees, 94th Cong., 2d Sess., 9 (Comm. Print 1976) (hereinafter Senate Report on Aging).
Congress included this provision in the original Act, and has retained it ever since. See Flemming v. Nestor, 363 U.S. 603, 610-611, 80 S.Ct. 1367, 1372-73, 4 L.Ed.2d 1435 (1960).
"The ten percent of workers not . . . covered by social security [in 1983] include[d] most Federal civilian workers (2.4 out of 2.7 million), about 30 percent of State and local employees (approximately 3 million), and 10-15 percent of employees of nonprofit organizations (up to 1 million)." H.R.Rep. No. 98-25, at 13, U.S.Code Cong. & Admin.News 1983, pp. 143, 231.
As Congress explained when it was studying the reasons underlying States' decisions to withdraw employees from the System, "[t]he Social Security Act of 1935 excluded from coverage all employment for States and localities, primarily because of the question of the constitutionality of any general levy of the employer tax on States and localities." Subcommittee on Social Security of House Committee on Ways and Means, Termination of Social Security Coverage for Employees of State and Local Governments and Nonprofit Groups, 97th Cong., 2d Sess., Ser. No. WMCP:97-34, p. 20 (Comm.Print 1982) (hereinafter H.R.Comm.Print 97-34).
At the time Congress enacted the amendment challenged in this case, it explained that provision for voluntary participation by employees of state and local governments was "the result of congressional desire to extend coverage as quickly and with as little difficulty as possible to those employees who needed it most." H.R.Rep. No. 98-25, at 19, U.S.Code Cong. & Admin.News 1983, p. 236.
Under the Act, therefore, States decide which groups of employees will receive Social Security coverage. Section 418(d)(3) creates an exception to this rule. Where state employees already are members of a retirement program, that section requires that a majority of such employees agree to participate in the Social Security System. 42 U.S.C. § 418(d)(3) (1982 ed. and Supp. II).
For purposes of conciseness, we use the term "Secretary" to refer to the federal official responsible for administration of the System both under the current and prior versions of the Social Security Act.
As of 1983, the employees of Alaska, "the only State to withdraw from the system, and of Maine, Massachusetts, Nevada, and Ohio, which never chose to participate in the system," were not covered by Social Security. H.R.Rep. No. 98-25, at 17, U.S.Code Cong. & Admin.News 1983, p. 235. Each of those States, however, was party to a § 418 Agreement that provided coverage to local government employees.
During these years, "many terminations were caused by consolidation of local jurisdictions, rather than by withdrawal from the social security system." Id., at 18, U.S.Code Cong. & Admin.News 1983, p. 236.
The Senate Special Committee on Aging found that States offered the following reasons for terminating their § 418 Agreements: employees wanted more take-home pay through a reduction in payroll deductions; state and local governments sought to cut costs by dropping Social Security coverage; news reports concerning "the projected exhaustion of social security trust funds in the 1980's" led employees to believe that benefits would cease; state and local governments believed that Social Security taxes would continue to rise, and thus viewed termination as a means "to achieve more static and budgetable expenditures"; some employees favored termination because they perceived that they would receive Social Security benefits even if they were no longer required to pay into the System; and alternative retirement plans were believed to pay higher levels of benefits. Senate Report on Aging 6-8; see also H.R.Comm.Print 97-34, at 6-7. The Committee also found that "many" decisions to withdraw from the System were made in the absence of "[i]nformation necessary for informed judgments." Senate Report on Aging 8.
Congress regarded voluntary participation by some employees, such as those of state and local governments, as an anomaly in an otherwise mandatory program. "The fundamental principle underlying compulsory coverage for most workers is that responsibility for paying for insurance against such risks should be borne by the society as a whole to the extent possible." H.R.Comm.Print 97-34, at 4. Mandatory participation is necessary to sustain the " 'pay-as-you-go' financing structure," particularly since workers retain coverage "regardless of how many times they change their jobs in a lifetime." Ibid. Mandatory participation ensures workers that they will obtain a minimum level of benefits in the event of a catastrophe that the worker did not foresee or plan for. Ibid. In the context of a mandatory system, voluntary participation for some employees was, in Congress' view, "inconsistent with the principle of equal treatment of all citizens." Id., at 5.
"(b) The amendment made by subsection (a) shall apply to any agreement in effect under [§ 418] on the date of the enactment of this Act, without regard to whether a notice of termination is in effect on such date, and to any agreement or modification thereof which may become effective under such [§ 418] after that date."
"The State, upon giving at least two years' advance notice in writing to the [Secretary], may terminate this agreement, either in its entirety or with respect to any coverage group, effective at the end of a calendar quarter specified in the notice, provided, however, that the agreement may be terminated in its entirety only if it has been in effect not less than five years prior to receipt of such notice, and provided further that the agreement may be terminated with respect to any coverage group only if it has been in effect with respect to such coverage group for not less than five years prior to receipt of such notice." Reprinted, App. 31.
If these employees were withdrawn from the System, "approximately $33.7 millio[n] would be lost to the social security trust funds in 1984." Id., at 61.
Plaintiffs in this lawsuit also alleged that the enactment denied them their contract rights without due process, that it constituted an attempt to regulate " 'essential state and local government functions,' in violation of the Tenth Amendment," and that they were entitled to specific performance for the Government's breach of contract. Public Agencies Opposed to Social Security Entrapment v. Heckler, 613 F.Supp. 558, 565 (ED Cal.1985).
Though the State did not press any claim that amended § 418(g) effected a taking of its property without the compensation required by the Fifth Amendment, the District Court rested its decision on that ground, finding it unnecessary to reach any of the arguments raised by the State. Therefore, none of those arguments are before us.
Before reaching the merits, the District Court determined that the plaintiffs in both suits, appellees here, had standing to challenge the validity of the enactment. With respect to the first lawsuit, brought by the public agencies and certain individuals, the court concluded that the agencies alleged an injury sufficient to confer standing because they claimed that amended § 418(g) deprived them of their contractual rights as third-party beneficiaries of the State's § 418 Agreement. Id., at 567-570. The individual plaintiffs had standing because they claimed that the federal defendants had denied them equal protection. Id., at 571. With respect to the second suit, the court found that the State had standing because it alleged "a judicially cognizable interest in the preservation of its own sovereignty, and a diminishment of that sovereignty by the alleged interference in its employment relations with its public employees." Id., at 567. While appellants suggest in this Court that none of the plaintiffs in the lawsuit brought by the public agencies were properly before the District Court, they concede, and we agree, that there is no question concerning the State's standing to bring the action. Therefore, the District Court plainly had authority to resolve this controversy, as do we.
Title 42 U.S.C. § 1304 provides: "The right to alter, amend, or repeal any provision of this chapter is hereby reserved to the Congress."
The language of § 418 and of California's § 418 Agreement provides further support for this conclusion. Section 418(a)(1) requires that the provisions of § 418 Agreements be "not inconsistent with the provisions of this section," 42 U.S.C. § 418(a)(1) (1982 ed., Supp. II), and the Agreement provided that it was "in conformity with" § 418. The State was thus on notice that the terms of its Agreement must mirror the provisions of the section, which could be amended under the reserved power. If Congress amended § 418 in such a manner as to render a provision of an Agreement "inconsistent" or no longer "in conformity" with the section, then the logical conclusion is that the inconsistent provision no longer was to be given legal effect.


text:
Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1α, 1β, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2α (but not R2β or R2γ) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-α release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.
Mast cells are ubiquitous in the body and are critical for allergic reactions due to their secretion of numerous vasoactive molecules and cytokines (1). Increasing evidence indicates that mast cells are also involved in innate and acquired immunity (2), as well as in neuroinflammatory conditions such as those affected by stress (3); these include asthma, arthritis, atopic dermatitis, and multiple sclerosis. Stress stimulates the hypothalamic-pituitary-adrenal axis through the release of corticotropin-releasing hormone (CRH),4 which activates behavioral, autonomic and endocrine responses, leading to secretion of glucocorticoids that down-regulate immune responses (4). CRH, however, is also secreted outside the brain where it exerts proinflammatory effects of stress apparently mediated through mast cells (5), possibly through direct activation of CRH receptors (6).
CRH exerts its effects by binding to specific cell surface receptors, of which two receptor subtypes, CRH-R1 and CRH-R2, have been characterized in human (7, 8), rat (9), and mouse (10). CRH-R1 and CRH-R2 share high sequence homology and belong to the family of seven transmembrane G protein-coupled receptors, stimulation of which activates adenylate cyclase, resulting in elevation of cAMP levels (11). A number of isoforms of the human CRH-R1 (1α, 1β, 1c, 1d, 1e, 1f, and 1g; Refs.7 , 12 , and 13) and three of the human CRH-R2 (2α, 2β, and 2γ; Refs.14, 15, 16) have been described, each containing different numbers of exons. Both receptor subtypes differ in tissue distribution: CRH-R1 predominates in brain, pituitary and skin, whereas CRH-R2 is present in brain, heart, and skeletal muscle (17). Binding sites for CRH have been reported on adherent splenocytes (18), T lymphocytes (19), and monocyte-macrophages (20). CRH-R1 and CRH-R2 expression was reported in granulocytes and lymphocytes, especially in inflamed s.c. tissues (21). CRH-R1 mRNA expression had been reported in the human leukemic mast cell line (HMC-1) (5). Mast cells in inflamed human synovial tissue were reported to express immunoreactive CRH-R1 (22). However, the expression of CRH receptor subtypes and their isoforms on mast cells are still poorly characterized. Moreover, no direct effect of CRH on human mast cells has been documented to date.
Acute restraint stress was shown to induce skin mast cell activation, an effect inhibited by a CRH receptor antagonist and the “mast cell stabilizer” sodium cromoglycate (cromolyn) (23). CRH also activated skin mast cells and induced mast cell-dependent vascular permeability in rodents (5). In addition, it was shown that i.p. CRH administration reproduced stress-induced colonic epithelial ion permeability changes in rats (24), whereas i.v. or intracerebral injection of CRH in nonstressed rats reproduced stress-induced colonic mucus release (25); these responses in both systems were blocked by a CRH receptor antagonist and the mast cell stabilizers doxantrazole or lodoxamide. However, none of these findings were shown to be mediated by a direct effect of CRH on mast cells. Mast cell-derived molecules that could increase vascular permeability include the well-known granule-stored histamine and tryptase (26), which would have an immediate action, as well as the newly synthesized vascular endothelial growth factor (VEGF) (27, 28) that could participate in delayed responses. In addition to vasoactive mediators, mast cells also secrete numerous proinflammatory cytokines, such as IL-6, IL-8, and TNF-α (1, 2). Interestingly, human mast cells were recently shown to synthesize and secrete large amounts of CRH (29), suggesting this peptide could regulate mast cells through CRH receptor in an autocrine manner.
We show by RT-PCR for the first time that both HMC-1 cells and human umbilical cord blood-derived mast cells (hCBMC) express mRNA for multiple CRH-R1 isoforms, whereas the latter also express CRH-R2α. CRH-R1 and CRH-R2 protein expression were also detected by Western blot analysis. Moreover, CRH induces selective release only of newly synthesized VEGF through CRH-R1-dependent activation of adenylate cyclase and significant increase of intracellular cAMP.
CRH, urocortin II, and urocortin III, as well as antalarmin and astressin 2B were purchased from Phoenix Pharmaceuticals and were dissolved in dH2O on the same day of the experiments. Forskolin, 8-bromo-cAMP, isobutyl-1-methylxanthine (IBMX), PMA, calcimycin A23187, actinomycin D, and cycloheximide were purchased from Sigma-Aldrich. SQ22536 was purchased from Calbiochem-Novabiochem.
HMC-1 cells were kindly supplied by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) and were cultured in IMDM (Invitrogen Life Technologies) supplemented with 10% (v/v) bovine calf serum (HyClone Laboratories), 100 U/ml penicillin/streptomycin, and 2 μM α-thioglycerol. Cells were kept under a humidified atmosphere of air/CO2 (19:1) at 37°C.
Human cord blood was obtained from human placentas from normal vaginal deliveries in accordance with established institutional guidelines. hCBMC were derived by the culture of CD34+ progenitor cells as previously described (30) with minor modifications. Briefly, mononuclear cells were isolated by layering heparin-treated cord blood onto Lymphocyte Separation Medium (ICN Biomedicals/Cappel). CD34+ progenitor cells were isolated from mononuclear cells by selection of cells positive for the AC133 Ag (CD133+/CD34+) by magnetic cell sorting (Miltenyi Biotec). For the first 4 wk, CD34+ cells were cultured in IMDM supplemented with 0.55 μM 2-ME, 100 mg/L insulin-transferrin-selenium, 0.1% BSA (Sigma-Aldrich), 100 U/ml penicillin/streptomycin, 100 ng/ml human recombinant stem cell factor (Amgen), and 50 ng/ml IL-6 (Chemicon International) at 37°C in 5% CO2 balanced air. After 4 wk of culture, BSA and insulin-transferrin-selenium in the culture medium were substituted with 10% FBS (Invitrogen Life Technologies).
Human HaCaT keratinocytes and the human melanoma SKMEL188 cell line (13, 31) were obtained from Dr. A. Slominski (University of Tennessee, Memphis, TN), and were cultured according to donor protocol.
The purity of hCBMC in culture was determined by immunostaining for mast cell specific tryptase, as well as by May-Giemsa staining as previously described (30). In our cultures, by 12 wk 100% of the cells were identified as mast cell. The mouse anti-human mast cell tryptase mAb was purchased from Chemicon International.
Total RNA was prepared from HMC-1 and hCBMC (100% mast cell purity) collected by centrifugation and lysed in 1 ml TRIreagent (107 cells/ml; Sigma-Aldrich) according to vendor instructions. First-strand cDNA synthesis was performed from 2 μg of total RNA using 200 U/μl Moloney Murine Leukemia Virus Reverse Transcriptase, 50 pmol/μl random hexamer oligonucleotide primer, 40 U/μl RNaseOUT recombinant RNase inhibitor (Invitrogen Life Technologies) and 10 mM dNTP (Roche Diagnostics). The reaction (20 μl) was run at 75°C for 5 min, 23°C for 10 min, 42°C for 45 min, and inactivated by heating at 95°C for 10 min. A total of 2 μl cDNA was used for PCR amplification. Oligonucleotide primers of previously published sequences for CRH-R1 and CRH-R2 isoforms (Table I⇓) were used (13, 32). Positive controls included human brain pituitary cDNA and the human melanoma SKMEL188 cell line cDNA for the CRH-R1 isoforms (13), human brain cerebral cortex cDNA for CRH-R2α (8), and human brain hippocampus cDNA for CRH-R2β and CRH-R2γ (16). PCR was performed in 50-μl volume containing 2 μl of cDNA, 0.2 mM of each dNTP, 2.5 U of TaqDNA polymerase (Invitrogen Life Technologies) with 1× PCR buffer and 15 pmol of each primer. The conditions for amplification were as follows: denaturation at 94°C for 1 min, primer annealing at 61°C for 1 min, and extension at 72°C for 1 min. Each PCR sample underwent a 40-cycle amplification. PCR products generated were visualized on a 2% agarose gel, stained with ethidium bromide, and viewed on a UV light box.
Nested PCR was performed to detect different CRH-R1 isoforms. The specific oligonucleotide primers previously published (13) to amplify human CRH-R1 exons 2–7 and exons 9–14 are listed in Table II⇓. The products of the first PCR served as a template for the second round of amplification reaction. Two rounds of PCR were conducted successively in a 25-μl volume containing 2 μl of DNA, 0.2 mM each dNTP, 1.25 U of TaqDNA polymerase with 1× PCR buffer, and 20 pmol of each primer. The mixture was heated to 94°C for 2.5 min, and then amplified for 35 cycles: 94°C for 30 s (denaturation), 65°C for 45 s (annealing), and 72°C for 1 min (extension). PCR products generated were visualized on a 2% agarose gel, stained with ethidium bromide, and viewed on a UV light box.
Human brain pituitary, cerebral cortex, and hippocampus cDNA was purchased from BD Clontech.
hCBMC (100% mast cell purity), HMC-1, and HaCaT cells were collected, lysed, and prepared for Western blot analysis. Samples were resolved on 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were incubated with goat anti-human/rat/mouse CRH-R1 that specifically recognizes CRH-R1, and goat anti-human/rat/mouse CRH-R2 that specifically recognizes CRH-R2, respectively (Santa Cruz Biotechnology) at a concentration of 1/1000 overnight at 4°C. As a negative control, the primary Abs were preabsorbed with synthetic receptor blocking peptides (1 μM; Santa Cruz Biotechnology). Following stripping and washing, the membranes were incubated with bovine anti-goat HRP-conjugated serum (Santa Cruz Biotechnology) at a dilution of 1/10,000 and anti-biotin HRP-conjugated Ab at a dilution of 1/1000 for 1 h at room temperature. The protein bands were detected using ECL reagent (Bio-Rad).
HMC-1 (40 × 106 cells) or hCBMC (20 × 106 cells) were collected, washed once in PBS, and homogenized with Polytron homogenizer (Kinematica) in 500 μl of extraction buffer A (50 mM Tris, pH 7.4, 2 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin). The homogenates were then centrifuged at 500 × g for 10 min at 4°C. The supernatant (plasma membrane and cytosolic fractions) was removed and centrifuged at 100,000 × g for 60 min at 4°C and the resulting pellet (plasma membrane) was solubilized in the extraction buffer A containing 1% Triton X-100. The supernatant was collected as the cytosolic fraction. The pellet from the initial 500 × g spin (nuclear fraction) was resuspended in extraction buffer B (10 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin) and microcentrifuged at high speed for 10 min. Supernatant (nuclear fraction) was collected. Protein concentration of all fractions was determined by Bio-Rad protein assay. Equal amounts of protein were run on 10% SDS-PAGE as described.
HMC-1 or hCBMC were washed with culture medium and incubated (1.5 × 106 cells/ml) for 20 min at 37°C in stimulation buffer (IMDM containing 1 mM of the potent phosphodiesterase inhibitor IBMX and 10 mM MgCl2). After centrifugation, cells were resuspended in stimulation buffer and incubated with human CRH, urocortin II, or urocortin III (100 nM) at 37°C for the indicated time points. The single concentration of 100 nM was chosen because preliminary results (data not shown) indicated that it induced the strongest response. The reaction was terminated by addition of 0.3 M HCl and the cells were frozen-thawed twice to release cAMP. The amount of cAMP released from the HMC-1 and hCBMC was estimated using a RIA kit (Biomedical Technologies).
For histamine and tryptase measurements, hCBMC were sensitized with human myeloma IgE for 48 h in culture medium and were then stimulated for 30 min in Tyrode’s buffer (133 mM NaCl, 4 mM KCl, 0.64 mM KH2PO4, 10 mM HEPES, 1 g/L glucose, 1 mM CaCl2, 0.6 mM MgCl2, and 0.03% human serum albumin, pH 7.2). Tryptase was measured in the supernatant by fluoroenzyme-immunoassay (Amersham Pharmacia Biotech). Histamine levels in the supernatant and pellet were measured using a LS-5B Luminescence Spectrometer (PerkinElmer) as described (33). Histamine release was expressed as percentage of the total cellular histamine content = ((histamine in supernatant)/(histamine in supernatant + histamine in pellet)) × 100.
For stimulation of cytokine production, HMC-1 (2 × 105 cells/200 μl) and hCBMC (105 cells/200 μl) were distributed to 96-well microtiter assay plates in duplicate or triplicate and stimulated in complete culture medium for the indicated concentrations of CRH. Optimal concentrations were determined from the dose-response experiments. For determination of whether secreted VEGF was de novo synthesized, cells were pretreated with the inhibitor of transcription actinomycin D (8 μM; Sigma-Aldrich) or the inhibitor of protein synthesis cycloheximide (50 μM; Sigma-Aldrich) for 30 min at 37°C before stimulation with CRH.
Cytokines were determined in cell-free supernatants with commercial ELISA kit (R&D Systems) according to the manufacturer’s directions. Cytokine secretion data were expressed as picograms per 106 cells.
The data are represented as the mean ± SEM from three or more experiments, each in duplicate or triplicate. For hCBMC, three or more separate experiments were done using mast cells derived from three or more different cord blood donors. Data were analyzed with the Student’s t test, Mann-Whitney U test, and ANOVA followed by the Holm-Sidak test or Newman-Keuls test as appropriate. Statistical significance was considered for p < 0.05.
To determine the purity of hCBMC, we conducted immunohistochemistry staining for the specific mast cell granule-stored protease, tryptase. In our cultures, by 12 wk 100% of the cells were identified as mast cells (data not shown). These hCBMC (12–16 wk of culture with 100% mast cell purity) were used for our study.
RT-PCR analysis showed that HMC-1 cells express specific mRNA for multiple CRH-R1 isoforms, including CRH-R1α, 1β, 1c, 1e, 1f, and 1g (Fig. 1⇓, a–c). CRH-R1α was the most abundant, 1c and 1e were prominent, whereas 1β, 1f, and 1g were faint. Similar studies in hCBMC showed that CRH-R1α, 1β, 1c, 1e, and 1f were present; CRH-R1g was absent (Fig. 1⇓, d–f). CRH-R1d was not detected in either HMC-1 or hCBMC (data not shown). Positive control used for CRH-R1d was the human melanoma SKMEL188 cell line that only expresses CRH-R1d.
RT-PCR and nested PCR analysis of CRH-R1 isoforms mRNA in HMC-1 cells (a–c) and hCBMC (d–f). Total RNA was extracted from HMC-1 cells and hCBMC and subjected to RT-PCR (a and d) and nested PCR (b, c, e, and f) analysis. Positive controls used commercially available cDNA. a and d, Human brain pituitary positive control for R1α (lane 1) and human mast cells (lane 2); human brain pituitary positive control for R1β (lane 3) and human mast cells (lane 4); and negative control (reaction mixture without DNA template) (lane 5) are shown. M, DNA size marker. b and e, Nested PCR amplification of human CRH-R1 exons 2–7. Human brain pituitary positive control for R1α, R1c, and R1e (lane 1) and human mast cells (lane 2) are shown. c and f, Nested PCR amplification of human CRH-R1 exons 9–14. Human brain pituitary positive control for R1α, R1f, and R1g (lane 1) and human mast cells (lane 2) are shown.
RT-PCR for CRH-R2 isoforms showed that HMC-1 cells do not express any (Fig. 2⇓, a–c), whereas hCBMC expressed only CRH-R2α (Fig. 2⇓, d–f). The PCR products were purified using QIAQuick PCR purification kit (Qiagen) and sequenced forward and reverse on ABI 3100 DNA sequencer at Tufts University Core Facility (Boston, MA). The sequencing results confirmed the identity and specificity of the PCR products.
RT-PCR analysis of CRH-R2 isoforms mRNA in HMC-1 cells (a–c) and hCBMC (d–f). Total RNA was extracted from HMC-1 cells and hCBMC and subjected to RT-PCR analysis. Positive controls used were commercially available cDNA. Human brain cerebral cortex cDNA was used as positive control for CRH-R2α; human brain hippocampus cDNA was used as positive control for CRH-R2β and CRH-R2γ. Positive control (lane 1), human mast cells (lane 2), and negative control (lane 3) are shown.
PCR amplification products using primers for β-actin as a housekeeping gene did not show any variation in the cDNA content among these samples; negative controls in which the reverse transcriptase was omitted to exclude any nuclear DNA contamination, or the reaction mixture without DNA template to exclude any system contamination, did not show any amplification product (data not shown).
Immunoblotting analysis was used to detect CRH receptor protein expression in HMC-1 cells and hCBMC. SDS-PAGE of whole cell lysates from both cell types was performed using Abs that specifically recognize CRH-R1 or CRH-R2. Whole cell lysate from HaCaT cells, an immortalized human keratinocyte cell line that is known to express CRH-R1 (34), was used as a positive control. The Ab to CRH-R1 yielded a predominant band of ∼45 kDa in both HMC-1 and hCBMC and another band of ∼70 kDa in HMC-1. A single band of 47 kDa was detected in HaCaT cells as reported previously (34) (Fig. 3⇓a). The specificity of the response was confirmed by preincubation of the CRH-R1 Ab with the CRH-R1 blocking peptide. Upon preabsorption with this peptide, the bands in all samples disappeared, confirming that the bands were specific for the CRH-R1 (Fig. 3⇓b). Immunoblotting for CRH-R2 identified a strong band of 49 kDa and a minor band of ∼59 kDa only in hCBMC (Fig. 3⇓c), which disappeared after pretreatment with the CRH-R2 blocking peptide (Fig. 3⇓d).
Western blot analysis of CRH-R1 (a and b) and CRH-R2 (c and d) in HMC-1 cells and hCBMC. hCBMC, HMC-1, and HaCaT cells were collected, lysed and samples of the whole cell lysates were run on 10% SDS-PAGE. hCBMC (lane 1), HMC-1 cells (lane 2), and HaCaT cells (lane 3) are shown. b and d, Membranes were stripped and the membrane proteins were analyzed with the anti-CRH-R1 Ab preabsorbed with the CRH-R1 blocking peptide (b) and the anti-CRH-R2 Ab preabsorbed with the CRH-R2 blocking peptide (d). HMC-1 (e) and hCBMC (f and g) were collected and membrane, cytosolic, and nuclear fraction were prepared as described in Materials and Methods. Equal amounts of protein were run on 10% SDS-PAGE. Membrane fraction (lane 1), cytosolic fraction (lane 2), and nuclear fraction (lane 3) are shown. CRH-R1 was detected in membrane fractions of HMC-1 (e) and h CBMC (f); CRH-R2 was detected in membrane fraction of hCBMC (g). MW, molecular weight marker.
CRH receptors belong to the family of seven transmembrane G protein-coupled receptors (11). We proceeded to examine whether CRH-R1 and CRH-R2 proteins are present in the membranes of human mast cells. Membrane, cytosolic, and nuclear fractions of the cells were collected, and equal amounts of protein from all fractions were analyzed by SDS-PAGE. Again, CRH-R1 was detected only in the membrane fractions of both cell types as a 45-kDa protein (Fig. 3⇑e, HMC-1 and Fig. 3⇑f, hCBMC) with another band of ∼70 kDa in HMC-1 cells. CRH-R2 was detected as a 49 kDa protein with a minor band at ∼59 kDa only in the membrane fraction of hCBMC (Fig. 3⇑g). The specificity of the response was again confirmed by preincubation of the CRH-R1 or CRH-R2 Ab with the corresponding blocking peptide (data not shown). The results suggest that CRH-R1 and CRH-R2 are membrane receptors in human mast cells.
CRH has been shown to exert its effects through binding to CRH receptor and activation of adenylate cyclase. To investigate whether the CRH-R1 on HMC-1 and hCBMC or CRH-R2 on hCBMC is functional, cAMP production was measured following stimulation with CRH. Incubation of HMC-1 cells with CRH (100 nM) increased intracellular cAMP significantly by 1.75 ± 0.12-fold (n = 6, p < 0.05) at 1 min, with a maximum of 2.0 ± 0.07-fold (n = 6, p < 0.05) at 3 min; thereafter, the cAMP levels decreased slowly, but still remained above basal values up to 1 h (Fig. 4⇓a). The level of cAMP in forskolin-treated cells increased from 3.0 ± 0.45-fold at 1 min to 15.0 ± 0.32-fold at 30 min (n = 6, p < 0.05) (data not shown), indicating that the adenylate cyclase system in cells was intact. Pretreatment of HMC-1 for 10 min with the selective CRH-R1 antagonist antalarmin (1 μM) significantly reduced cAMP to basal levels (n = 6, p < 0.05), whereas the selective CRH-R2 antagonist astressin 2B (1 μM) did not have any effect on cAMP (Fig. 4⇓b); there was no effect even at higher concentrations (5–100 μM) (data not shown), confirming that CRH-R2 is not present in HMC-1 cells and indicating that CRH-induced cAMP increase was mediated through CRH-R1.
Effect of CRH on cAMP production in HMC-1 cells and hCBMC. a and c–e, Cells were stimulated with either CRH or urocortin (Ucn II or Ucn III) (100 nM) in serum-free stimulation buffer, and intracellular cAMP levels were then measured by specific RIA at the indicated time points. b, Pretreatment of HMC-1 cells for 10 min with the selective CRH receptor antagonists followed by CRH (100 nM) stimulation. f, Pretreatment of hCBMC for 10 min with the selective CRH receptor antagonists (1 μM) followed by CRH (100 nM) stimulation. Ant, antalarmin; Ast, astressin 2B. Data are the mean ± SEM (n = 6). ∗, p < 0.05 vs control, ∗∗, p < 0.05 vs CRH.
Similar studies were conducted in hCBMC, but due to the shortage of the cells, cAMP levels were only measured at 1 and 3 min. Incubation of hCBMC with 100 nM CRH (Fig. 4⇑c), as well as the specific CRH-R2 agonists urocortin II (Fig. 4⇑d) or urocortin III (Fig. 4⇑e) increased cAMP significantly (n = 6, p < 0.05) at 3 min. Pretreatment of hCBMC for 10 min with antalarmin (1 μM) reduced the effect of CRH on cAMP increase to basal levels (Fig. 4⇑f), whereas astressin 2B (1 μM) completely blocked the effects of urocortin II/III (data not shown). In contrast, pretreatment of cells with astressin 2B (1 μM) had no effect on CRH-induced cAMP elevation (Fig. 4⇑f), not even at higher concentrations (5–100 μM) (data not shown), indicating that cAMP could be selectively increased by CRH through CRH-R1. The antagonists alone at the concentrations used did not alter basal cAMP levels in either HMC-1 or hCBMC.
Stimulation of the cells with CRH caused a dose-dependent (24 h incubation) and time-dependent release of VEGF from both HMC-1 (Fig. 5⇓, a and b) and hCBMC (Fig. 5⇓, c and d). Secretion was blocked by preincubating cells with antalarmin (1 μM), but not with astressin 2B (1 μM) (Fig. 5⇓, e and f). Higher concentrations of astressin 2B (5–100 μM) also had no effect. HMC-1 cells treated with PMA (25 ng/ml) plus calcium ionophore A23187 (250 nM), as a positive control for VEGF release, secreted 4540.2 ± 220.6 pg of VEGF per 106 cells (n = 6), ∼20-fold increase compared with the untreated control. Human myeloma IgE-sensitized hCBMC for 24 h challenged with anti-IgE for another 24 h as a positive control, released 88.9 ± 2.1 pg of VEGF per 106 cells (n = 6), ∼27% increase in comparison with the untreated control (data not shown). The delayed time course of VEGF secretion (6 h earliest increase) suggested that CRH stimulated de novo synthesis of VEGF. Indeed, VEGF production was completely inhibited by pretreatment (30 min) with the inhibitor of protein synthesis, cycloheximide (50 μM), in both HMC-1 (Fig. 5⇓b) (100% inhibition; n = 3) and hCBMC (Fig. 5⇓d) (100% inhibition; n = 3). Similar results were obtained using the inhibitor of transcription, actinomycin D (8 μM) (data not shown). Taken together, these results suggest that CRH selectively activated CRH-R1 for VEGF release in both HMC-1 and hCBMC, and VEGF was synthesized de novo.
CRH stimulates VEGF secretion in HMC-1 cells and hCBMC. Dose-dependent secretion of VEGF in HMC-1 cells (a) and hCBMC (c). Cells were plated in 96-well plates and stimulated with CRH (24 h incubation) in a dose-dependent manner in complete culture media. Time-dependent secretion of VEGF in HMC-1 cells (b) and hCBMC (d). Cells were plated in 96-well plates and stimulated with CRH in a time-dependent manner (CRH 100 nM for HMC-1 and 1 μM for hCBMC) in complete culture media. Additionally, the CRH-stimulated and the unstimulated release of VEGF was monitored in the presence of 50 μM cycloheximide. Pretreatment of cells with antalarmin (1 μM) or astressin 2B (1 μM) followed by CRH (100 nM) stimulation for 24 h in HMC-1 (e) and hCBMC (f). Ant, antalarmin; Ast, astressin 2B. Data are the mean ± SEM (n = 6). ∗, p < 0.05 vs control, ∗∗, p < 0.05 vs CRH.
We next investigated whether VEGF secretion was accompanied by tryptase and histamine release, which are two of the major granule-stored mediators in mast cells. hCBMC were stimulated with CRH at 100 nM for 30 min. Tryptase and histamine secretion in response to stimulation with anti-IgE (10 μg/ml) was used as a positive control. Tryptase and histamine secretion was similar between untreated control and CRH-treated cells (Fig. 6⇓, a and b). Similar results were obtained from HMC-1 cells, using PMA (25 ng/ml) plus calcium ionophore A23187 (0.5 μg/ml) treated cells as a positive control (data not shown). These results were further confirmed in both cells treated with different concentrations of CRH and by extending the time period to 3, 6, and 24 h for tryptase and histamine release measurements (data not shown). Moreover, there was no release of IL-6, IL-8, or TNF-α under these conditions in both hCBMC (Fig. 6⇓, c–e; note that the SEM for IL-6 values was large in this cell type) and HMC-1 cells (Fig. 6⇓, f–h). In addition, CRH did not enhance the effect of anti-IgE on mediator release in hCBMC (Fig. 6⇓, a–e) or the effect of IL-1 or PMA plus calcium ionophore on mediator release in HMC-1 (Fig. 6⇓, f–h). These data demonstrate that CRH induced selective release of VEGF in human mast cells in the absence of concomitant tryptase and histamine secretion and IL-6, IL-8, or TNF-α release.
CRH selectively stimulates production of VEGF without tryptase, histamine, IL-6, IL-8, and TNF-α release. Tryptase (a) and histamine (b) secretion by hCBMC in response to stimulation with anti-IgE (10 μg/ml) in the presence or absence of CRH (100 nM). Cells were stimulated for 30 min for tryptase and histamine measurements. IL-6 (c), IL-8 (d), and TNF-α (e) release by hCBMC in response to stimulation with anti-IgE (10 μg/ml) in the presence or absence of CRH (100 nM) for 6 h. IL-6 (f), IL-8 (g), and TNF-α (h) release by HMC-1 cells in response to stimulation with IL-1 (10 ng/ml) or PMA (25 ng/ml) plus A23187 (0.5 μg/ml) in the presence or absence of CRH (100 nM) for 6 h. P, PMA; A, A23187. Data are the mean ± SEM (n = 4). ∗, p < 0.05 vs control.
CRH binding to CRH-R1 typically activates adenylate cyclase leading to increased intracellular concentrations of cAMP (11). Because we showed that CRH-stimulated VEGF release was CRH-R1-dependent and CRH significantly increased cAMP in both HMC-1 and hCBMC, we proceeded to investigate whether cAMP is involved in CRH-mediated VEGF production. Forskolin, a direct activator of adenylate cyclase, and 8-bromo-cAMP, a cell-permeable cAMP analog mimicked the effect of CRH and induced strong VEGF secretion in both HMC-1 (Fig. 7⇓, a and b) and hCBMC (Fig. 7⇓, c and d) in a dose-dependent manner. We then stimulated cells with CRH in the presence or absence of SQ22536, an adenylate cyclase inhibitor. The effect of CRH on VEGF production was completely abolished with SQ22536 at the concentration of 8 mM (Fig. 7⇓, e and f). SQ22536 at this concentration did not alter the basal level of VEGF production in both cells. HMC-1 cells treated with forskolin (1 μM) for 6 h and hCBMC treated with forskolin (10 μM) for 24 h were used as positive controls for adenylate cyclase activation. SQ22536 (8 mM) significantly reduced forskolin-induced VEGF release in HMC-1 cells from 334.1 ± 45.9 pg per 106 cells to 54.8 ± 3.6 pg per 106 cells, comparable to untreated control (n = 4, p < 0.05). Similar results were observed in hCBMC in which VEGF production was significantly reduced from 119.4 ± 7.8 pg per 106 cells to 71.6 ± 2.5 pg per 106 cells (n = 4, p < 0.05). Our results strongly suggest that CRH stimulates VEGF production in human mast cells through CRH-R1-dependent activation of adenylate cyclase and increased cAMP.
CRH-stimulated VEGF production is mediated through cAMP. Dose-dependent secretion of VEGF in HMC-1 cells (a and b) and hCBMC (c and d) in response to forskolin (a and c) and 8-bromo-cAMP (b and d). Cells were plated in 96-well plates and stimulated with forskolin or 8-bromo-cAMP as indicated in a dose-dependent manner (6 h incubation for HMC-1 and 24 h incubation for hCBMC) in complete culture media. The effect of SQ22536 on CRH-induced VEGF production in HMC-1 cells (e) and hCBMC (f) is shown. Cells were plated in 96-well plates and preincubated with SQ22536 (8 mM) for 1 h before stimulation with CRH (100 nM for HMC-1 and 1 μM for hCBMC) for another 6 h (HMC-1) or 24 h (hCBMC) in complete culture media. Data are the mean ± SEM (n = 4). ∗, p < 0.05 vs control, ∗∗, p < 0.05 vs CRH.
To our knowledge, this is the first report that mRNA of multiple CRH receptor isoforms is expressed in human mast cells. Moreover, CRH-R1 protein is present and functional in HMC-1, whereas both CRH-R1 and CRH-R2 are functional in hCBMC, as evidenced by a significant increase of cAMP production induced by CRH or the specific CRH-R2 agonists urocortin II or urocortin III, respectively. This is also the first time that CRH is shown to stimulate selective secretion of VEGF without tryptase and histamine or cytokines IL-6, IL-8, or TNF-α. The SEM of IL-6 release in hCBMC was fairly large, possibly due to different batches of hCBMC from separate donors. Both cAMP and VEGF production induced by CRH were blocked by pretreatment with the specific CRH-R1 antagonist antalarmin, but not the specific CRH-R2 antagonist astressin 2B, indicating they were mediated through CRH-R1. Moreover, CRH activated adenylate cyclase, leading to an increase of cAMP that mediated VEGF production. The selective VEGF release could explain how mast cells participate in inflammatory processes in the absence of allergic stimulation or degranulation. Mouse and human mast cells had previously been shown to secrete both preformed and newly synthesized VEGF in response to allergic stimulation (27). Human leukemic and skin mast cells were also shown to constitutively express VEGF and could also further express VEGF 206, a vasodilatory isoform, upon prolonged stimulation (28).
CRH-R1 mRNA expression had previously been reported in HMC-1, but was not characterized further (5). In this study we showed that in both HMC-1 and hCBMC, CRH-R1α is the most abundant, CRH-R1c and CRH-R1e were prominent, with CRH-R1β and CRH-R1f being faint. The CRH-R1d isoform was absent in both HMC-1 and hCBMC, whereas CRH-R1g was absent only in the latter. The lack of isoform-specific Abs did not allow us to test which of these receptor isoform proteins is expressed in HMC-1 or hCBMC. No function has so far been attributed to these isoforms in mast cells. The gene for human CRH-R1 contains 14 exons. Several splice variants of the mRNA for this receptor have been identified, encoding different isoforms, termed R1α, 1β, 1c, 1d, 1e, 1f, and 1g. CRH-R1β contains all 14 exons translating into a 444 amino acid protein, 1α lacks exon 6, 1c lacks exons 3 and 6, whereas 1d lacks exons 6 and 13. Exons 3 and 4 were spliced from CRH-R1e; exon 12 was spliced from CRH-R1f; exon 1, and 27 bp of exon 10 and 28 bp of exon 12 were spliced from CRH-R1g (13). The major ligand-binding determinant of mammalian CRH-R1 has been mapped to its first extracellular domain, coded by exons 1–4 of CRH-R1 (35). Exon 3 contains two regions that are critical for high-affinity ligand binding, and mutations in this region abolish CRH binding (36). CRH-R1α, 1f, and 1g encode the entire CRH-binding domain; they should, therefore, be the efficient receptor variants that bind and transduce peptide signals into cAMP-mediated pathways. In contrast, isoforms R1β, R1c, and R1e either have decreased ligand-binding capacity (β, c) or are poorly coupled to cAMP production (β, e) (36, 37, 38). COS cells were recently transfected with different CRH-R1 isoforms (39). It was shown that CRH-R1α was the only isoform coupled directly to adenylate cyclase, whereas CRH-R1g inhibited the activity of the cyclase regulatory element (39). Hence, different CRH receptor isoforms could link to different signaling pathways.
Based on the amino acid sequence of CRH receptors, the predicted molecular mass values range between 45 and 53 kDa (40). Indeed, the CRH-R1 protein we detected by Western blot analysis in both HMC-1 and hCBMC is ∼45 kDa, and the CRH-R2 protein in hCBMC is ∼49 kDa. A single band of 47 kDa for CRH-R1 was detected in the HaCaT cells sample, consistent with a previous report (34). High molecular mass bands for CRH-R1 (∼70 kDa) and CRH-R2 (∼59 kDa) were detected in HMC-1 cells and hCBMC, respectively. These high molecular mass bands could be attributable to posttranslational modifications of the CRH receptors. Previous studies using immunoblotting analysis also showed that CRH receptors in human placenta had molecular mass of 55 kDa and 75 kDa (41, 42, 43). CRH-R1 of 59 and 72 kDa were identified in the rat and mouse pituitary membranes (44). In addition, the molecular weights of rat and mouse brain CRH-R1 were determined by Western blot analysis to be 80,000–76,000 and 83,000–79,000, respectively (44). AtT20 mouse pituitary tumor cells were reported to express CRH-R1 of 70 kDa (45). The different molecular mass values of the CRH receptor molecules point to a different regulation of the receptor modification in various tissues and cell types. Due to differences in the posttranslational modification of the proteins, the molecular mass of CRH receptors could differ from one tissue or cell type to another. CRH receptors belong to the family of seven transmembrane G protein-coupled receptors. Using Western blot analysis, we showed that CRH-R1 and CRH-R2 are membrane receptors in both cells because the receptors are present only in the membrane fractions of these cells.
Our present findings showing selective VEGF release are seemingly different from the intradermal injection of CRH (5) that was shown to degranulate skin mast cells and increase vascular permeability. This apparent in vitro and in vivo discrepancy could be explained by preliminary evidence indicating that the intradermal effect of CRH leading to mast cell degranulation may involve the synergic effect of neurotensin (NT) (our unpublished observation) because pretreatment with an NT receptor antagonist blocked the intradermal effect of CRH (6). In other words, intradermal CRH may have two effects: 1) induction of VEGF release by a direct action on skin mast cells, and 2) induction of skin mast cell degranulation by stimulating release of NT from dorsal root ganglia nerve processes, which in turn stimulates mast cell degranulation. Our preliminary findings indicate that mouse dorsal root ganglia express NT (our unpublished observations), whereas receptors for NT were recently identified on rat mast cells (46).
Our results showed that CRH-induced VEGF production by human mast cells is mediated through an increased intracellular cAMP because the direct adenylate cyclase activator forskolin and cell-permeable cAMP analog 8-bromo-cAMP mimicked the effect of CRH, and the adenylate cyclase inhibitor SQ22536 completely abolished CRH-induced VEGF production. Previous findings by other groups also suggested involvement of cAMP in VEGF production. For example, salbutamol, a β2-adrenoceptor agonist widely used as a bronchodilator in the treatment of asthma and known to elevate cAMP levels, induced an increase in VEGF production by hCBMC in a dose-dependent manner, as did forskolin (47). In addition, cAMP-elevating agents have been shown to stimulate VEGF production in macrophages (48). In contrast, cAMP could also have an inhibitory influence on mediator release. For example, a specific β2-adrenoceptor agonist inhibited the IgE-mediated release of tryptase and TNF-α production in hCBMC (49). One way to possibly reconcile these data would be that cAMP could have divergent effects on different intracellular processes. It has previously been reported that cAMP may have both inhibitory and stimulatory effects in the same cell type (50). Such seemingly opposing effects could be due to pleiotropic actions of CREB proteins (51). Once activated, these proteins bind to cAMP response elements of various genes and act either as positive regulators or dominant-negative inhibitors of transcription. Because the complement of CREB proteins differs among cell types and their effects are dependent upon the stimulus, CREB activation can produce a number of apparently conflicting effects.
CRH profoundly influences neuroendocrine-immune interactions indirectly, through activation of the hypothalamic-pituitary-adrenal axis and release of glucocorticoids and catecholamines (4). Peripheral CRH secreted locally at inflammatory sites, however, could have autocrine or paracrine immunomodulatory actions (52), especially because mast cells secrete a large amount of CRH (29). Recent evidence, in fact, indicates that mast cells are involved in inflammatory diseases worsened by stress (3). For instance, mast cells are increased in psoriasis, the symptoms of which worsen by stress (53) and which has been associated with increased VEGF levels (54). Mast cells are also known to be involved in skin delayed hypersensitivity reactions (55) that are also associated with increased VEGF expression (56) and worsen by stress (53). Increased and activated mast cells have been documented in the bladder of patients with interstitial cystitis (57), an inflammatory condition recently associated with increased VEGF expression (58). Finally, mast cells are also increased (59) and express CRH-R1α in the joints of rheumatoid arthritis patients in which the expression of CRH-R1 and CRH-R2 mRNAs was detected by RT-PCR (23). Immunoreactive CRH (60) was also detected in the synovium of patients with rheumatoid arthritis reported to have increased VEGF (61). Inflammatory arthritis was also greatly reduced in CRH knockout mice (62), whereas collagen-induced arthritis was reduced by the CRH-R1 antagonist antalarmin (63).
Our results support the premise that CRH could either initiate or potentiate inflammatory processes (3) by acting directly on mast cells to release VEGF selectively and with a time course different from that of anaphylactic reactions. Ongoing investigations address the biological significance of this phenomenon under inflammatory conditions.
We thank Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) for kindly providing the HMC-1 cells, Dr. A. Slominski (University of Tennessee, Memphis, TN) for providing the HaCaT keratinocytes and human melanoma SKMEL188 cell line, and Amgen for the human recombinant stem cell factor. We are grateful to Dr. A. Athanassiou and the nurses of the Labor and Delivery Division, Tufts-New England Medical Center (Boston, MA), for umbilical cord blood collection. We also thank Jessica Christian for patience and word processing skills.
↵1 This work was supported in part by National Institutes of Health Grant AR47652 (to T.C.T.).
↵2 Current address: Division of Information Biology, Department of Biosystem Information Sciences, Tohoku University, Aramaki aza Aoba 09, Aoba-ku Sendai-city Miyagi-Pref., 980-8579, Japan.
↵4 Abbreviations used in this paper: CRH, corticotropin-releasing hormones; HMC-1, human leukemic mast cell line; hCBMC, human cord blood-derived mast cell; NT, neurotensin; VEGF, vascular endothelial growth factor.
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matches: V. V. V. V. V. V.


1. Should Claimant’s temporary total disability benefits have terminated either on February 1, 2012 or on August 19, 2012 on the grounds that his ongoing disability was causally related to a personal medical condition rather than to his compensable work injury?
2. Alternatively, should Claimant’s temporary total disability benefits have terminated on August 19, 2012 on the grounds that he had delayed seeking medical treatment for his compensable work injury without a reasonable basis for doing so?
1 The parties having agreed to submit this claim on briefs and written evidence only, Defendant’s Motion to Strike those portions of Claimant’s proposed Findings of Fact and Conclusions of Law that reference Dr. Abdu’s “expected” testimony is hereby GRANTED.
3. Claimant was the head of maintenance at Defendant’s automobile dealership. His duties were varied, and included both janitorial work and general maintenance.
4. On October 5, 2009 Claimant was at work, removing a pressure washer from the bed of a pickup truck. As he pulled on the washer, the hose got caught in the tailgate. Claimant felt a burning pain in his lower back, with radiating pain down his right leg.
5. Defendant accepted Claimant’s lower back injury as compensable, and began paying workers’ compensation benefits accordingly.
7. Claimant’s symptoms having failed to resolve with either physical therapy or injections, in June 2010 Ms. King referred him to Dr. Abdu, an orthopedic surgeon, for further evaluation. For a number of reasons, initially Dr. Abdu suggested additional conservative management and physical therapy rather than surgery. For that, he directed Claimant back to Ms. King. In the meantime, on a form questionnaire he indicated that Claimant had no work capacity as of the date of his examination.
8. Ms. King next evaluated Claimant in August 2010. Based on her examination, she concluded that modified duty work “should be tolerable;” however, she noted that were Claimant to consider vocational rehabilitation a functional capacity evaluation likely would be necessary. Thereafter, in September 2010 Ms. King’s practice partner, Dr. Hynninen, suggested that work hardening might be a consideration in the future, as apparently Defendant still had not been able to make modified duty work available.
9. Because his symptoms had not responded to further conservative management, in November 2010 Ms. King referred Claimant back to Dr. Abdu. This time, Dr. Abdu determined that surgical intervention was appropriate. Due to a number of factors, however, the procedure was not scheduled until mid-October 2011. In the interim, on form questionnaires completed in both May and October 2011 Dr. Abdu continued to indicate that Claimant was totally disabled from working as a result of his lower back injury.
10. Unfortunately, in early October 2011 Claimant began to experience symptoms indicative of a serious cardiac problem, including shortness of breath, chest tightness and atrial fibrillation, or rapid and irregular heartbeat. As a result, Dr. Abdu’s surgery had to be delayed indefinitely so that Claimant could undergo treatment for his heart condition.
11. From October 2011 through July 2012 Claimant treated regularly with Dr. Tepfer, a cardiologist. Dr. Tepfer diagnosed dilated cardiomyopathy, a condition in which the heart becomes enlarged and weakened to the point where it cannot pump blood efficiently. Cardiomyopathy can be treated with medications, but finding the correct drug combinations and dosages requires cautious medical management and frequent monitoring. In Claimant’s case, this process was further complicated by the fact that he continued to suffer from both atrial fibrillation and congestive heart failure. The latter condition occurs when the heart is not able to pump enough blood to the rest of the body.
12. As of the time he first evaluated Claimant in October 2011, Dr. Tepfer characterized his condition as severe and life-threatening. He could hardly walk and was in very great distress. While he made good progress with treatment over the ensuing months, his heart function remained abnormal and his physical capacity was diminished. I find from the medical evidence that while the disability referable to Claimant’s lower back injury persisted during this time, concurrently his heart condition rendered him totally disabled as well.
13. Dr. Tepfer last examined Claimant on July 27, 2012. On that date, he advised Dr. Bresnahan that Claimant’s heart condition had improved sufficiently for him to undergo back surgery, and that at least from a cardiac perspective he was no longer disabled. Because he was closing his practice, Dr. Tepfer arranged for Claimant to follow up with another cardiologist, Dr. Burke, thereafter. The medical records do not indicate whether or when Claimant did so.
14. As of the date the record closed, Claimant had not yet been scheduled for back surgery. He was to undergo a pre-operative visit with Dr. Abdu on December 5, 2012. The record does not indicate whether Dr. Abdu will require updated medical clearance from either Claimant’s current cardiologist and/or from his primary care physician prior to proceeding with surgery.
15. There is no evidence establishing that Dr. Abdu has at any time rescinded or otherwise amended his October 2011 determination that Claimant was totally disabled from working as a consequence of his work-related lower back injury. Nor has any other medical provider stated a contrary opinion since that time.
16. Defendant has twice sought to discontinue Claimant’s temporary disability benefits, once on February 1, 2012 and more recently on August 19, 2012. As grounds for the first discontinuance, Defendant alluded to the fact that Claimant’s heart condition was preventing him from undergoing treatment for his work-related lower back injury. As grounds for the second discontinuance, it stated that Claimant’s heart condition constituted an intervening event that had both delayed treatment for his work injury and further deteriorated his physical capabilities.2 The Department denied both discontinuances on the grounds that they were not reasonably supported.
1. The disputed issue in this case is whether the disability associated with Claimant’s non-work-related heart condition has at any time since February 2012 justified discontinuing the temporary disability benefits referable to his work-related lower back injury. Defendant having accepted and paid benefits on account of the latter injury, the burden is on it to prove that discontinuing those payments is proper. Merrill v. University of Vermont, 133 Vt. 101, 105 (1974); Luff v. Rent Way, Opinion No. 07-10WC (February 16, 2010).
2. The Vermont Supreme Court has considered the question whether temporary disability benefits can be suspended when treatment for a work-related injury is delayed on account of a non-work-related condition. In Wood v. Fletcher Allen Health Care, 169 Vt. 419 (1999), it held that a claimant who could not undergo surgery for a work-related shoulder injury because she was pregnant was entitled to ongoing disability payments notwithstanding that the period during which she remained unable to work was prolonged as a result. Noting the directive that Vermont’s workers’ compensation statute be liberally construed in favor of injured workers, Montgomery v. Brinver Corp., 142 Vt. 461, 463 (1983), the court concluded that the pregnancy was not a superseding, intervening event sufficient to break the causal link between the claimant’s work-related injury and her resulting disability. Id. at 424.
3. A key difference between the circumstances presented here and those described in Wood is that the non-work-related condition in the latter case – the claimant’s pregnancy – was not independently disabling.3 That is not the case here; rather, the undisputed evidence establishes that Claimant’s cardiomyopathy was, at least for a time, not just totally disabling but indeed life threatening. The question, therefore, is whether this fact justifies a different result.
2 In its proposed Findings of Fact and Conclusions of Law, Defendant asserts as an additional ground in support of its August 2012 discontinuance that Claimant has unreasonably delayed pursuing surgical treatment for his low back injury following clearance from his cardiologist to do so.
3 Likewise the other cases cited by Claimant in support of his position, Schock v. Morristown Memorial Hospital, Superior Court of New Jersey, Appellate Div. No. A-1658-09T2 (July 2, 2010), Thomas v. Burggraf Restoration, 31 P.3d 402 (Okla. 2001), and Workmen’s Compensation Appeal Board v. Chamberlain Manufacturing Corp., 336 A.2d 659 (Pa. 1975).
4. I conclude that it does not. As it was in Wood, the critical fact here is that during the time when Claimant’s non-work-related condition precluded treatment, his work-related injury continued to be totally disabling. Given the express language of the statute, which mandates that temporary total disability benefits be paid for so long as the work injury “causes total disability for work,” 21 V.S.A. §642, sufficient grounds for discontinuing them in this case do not yet exist.
5. Had there been credible evidence establishing that Claimant’s lower back injury ceased to be totally disabling at any time after Dr. Abdu’s October 2011 determination that it was, the facts might justify a different result. Without such evidence, to assert that Claimant’s heart condition rendered him even more totally disabled than he already was is a non sequitur.
6. I conclude that Defendant has failed to sustain its burden of proving that Claimant’s work-related disability had ended as of either February 1, 2012 or August 19, 2012. Its attempts to discontinue temporary total disability benefits on those dates were appropriately rejected, therefore.
7. As for whether Defendant should be allowed to terminate Claimant’s temporary disability benefits on the grounds that he has unreasonably delayed seeking surgical treatment since Dr. Tepfer cleared him to do so, I conclude that the evidence is similarly lacking. Although the medical records do not document any recent treatment, there is no way for me to know whether this is due to Claimant’s inaction, scheduling difficulties with his doctors or some combination of both. Under the circumstances of this case, particularly the serious nature of Claimant’s cardiac condition and the need for him to transfer his care to a new cardiologist, Defendant has failed to establish that a four-month delay in resuming treatment for his lower back condition is so egregious as to warrant a suspension of benefits.
8. I conclude that Defendant has failed to establish sufficient grounds to justify terminating Claimant’s temporary total disability benefits on either February 1, 2012 or August 19, 2012. Therefore, it is not entitled to any offset or credit against future wage replacement benefits to be paid on account of Claimant’s October 5, 2009 work-related injury.
9. As Claimant has prevailed, he is entitled to an award of costs and attorney fees. In accordance with 21 V.S.A. §678(e), Claimant shall have 30 days from the date of this opinion within which to submit his itemized claim.
Based on the foregoing findings of fact and conclusions of law, Defendant’s claim for a credit or offset against future wage replacement benefits for temporary total disability benefits paid since either February 1, 2012 or August 19, 2012 is hereby DENIED. Defendant is hereby ORDERED to pay Claimant’s costs and attorney fees as awarded in accordance with 21 V.S.A. §678.
DATED at Montpelier, Vermont this 3rd day of January 2013.