(Oregon. Health. Sciences. University) for gifts of antisera,. Drs. Evelyn. Kurt-Jones and. Abul. Abbas ... D. A., and Blalock,. J. E. (1987) Interactions between. 4.
Nonopiate
active
are secreted K. A. ROTH,’ Department
proenkephalin-derived by T helper
R. G. LORENZ,
of Pathology,
Washington
R. A. UNANUE, University
Recent investigations cnine and immune
have
shown
that
the neuroendo-
systems profoundly affect each other. In part, these interactions occur via common chemical messengers and receptors. One possible shared chemical messenger is the opioid precursor preproenkephalin, for which high concentrations of messenger RNA tivated T helper
are present in brain, adrenal, and accells. Because the biologic action of
most peptide messengers depends on the posttranslational processing of the precursor, we have examined T helper cell lines for the production of proenkephalinderived peptides. These peptides were characterized by multiple radioimmunoassays, gel filtration chromatography, and opiate radioreceptor assays. We found that activated T helper cells secrete significant concentrations
of high-molecular-weight,
opiate-inactive
tides, which are distinct from the derived peptides of the neuroendocrine studies clearly indicate enkephalin, and suggest
products tions.-
pep-
proenkephalinsystem. These
cell-specific processing of prothat the T helper cell-secreted
may have nonopiate
receptor-mediated
ac-
ROTH, K. A.; LORENZ, R. G.; UNANUE, R. A.; WEAVER, C. T Nonopiate active proenkephalin-derived peptides are secreted by T helper cells. FASEB J. 3: 2401-2407; 1989. Key
Words:
opioids
enkephalin
T
helper
cells
cells AND
C. T. WEAVER
School of Medicine,
ABSTRACT
post-
translationalprocessing
St. Louis,
in the produce
circulatory
can
be modulated by endocrine hormones, particularly corticosteroids (1), and data suggest that products secreted by immune cells, such as interleukin 1 (IL 1),2 affect neuroendocnine function (2). Some evidence indicates that neuroendocrine-immune interactions may occur via chemical messengers common to both neuroendocrine and immune cells (3). This last hypothesis is supported by the immunohistochemical localization of IL 1 in hypothalamic neurons (4), proopiomelanocortin (POMC) peptide products in rat splenic macrophages (5), and the presence of high levels of messenger RNA
0892-6638/89/0003-2401/$01
.50. © FASEB
system
that stimulated
abundant
yet the peptide have not been responses
Missouri
63110,
USA
for preproenkephalin in activated murine T helper cells (6, 7). Proenkephalin and POMC are precursors for the endogenous opiates (opioids), endorphins and enkephalins. Opioid peptides were first isolated from brain, pituitary, and adrenal extracts (8-10) and subsequently shown to arise from three opioid precursors: proenkephalin (11, 12), POMC (13), and prodynorphin (14). These three precursors contain 11 copies of the common opioid sequence Tyr-Gly-Gly-Phe-(Met or Leu). Posttrans]ational processing of the opioid precursors leads to the formation of more than 20 opiate receptor-active peptides (15). This processing is an important regulatory step in opioid biology, inasmuch as a free amino-terminal tyrosine in the common opioid sequence is essential for opiate receptor binding (16); the high-molecular-weight precursors in which the opioid sequences are embedded are themselves devoid of opiate activity. In addition, carboxyl-terminal extensions of the common opioid sequence determine opiate receptor subtype specificity and peptide stability (17, 18). In the neuroendocnine system, the type and extent of posttranslational processing is largely tissue specific. For example, in the brain, proenkephalin is extensively processed to yield low-molecular-weight (15,000 to about 3000. Free opioid material was present in =
=
=
=
E “I
1#{149}
0 E 0 0.
U
>I.-
4 DISCUSSION
> 1-
U
3
LU
0
2
z 20
40
60
80
00
120 140
TIME (hours) Figure
1. D10.G4.1 cells were stimulated
at time zero with Con A,
and the supernatants collected and extracted at the indicated times and examined by RIA for total opioids (peptides containing the sequence Tyr-Gly-Gly-Phe-X anywhere in their sequence) (#{149}), Metenkephalin-Arg-Phe (0), free opioids (peptides beginning with the sequence Tyr-Gly-Gly-Phe-X) (A), and Met-enkephalin (s).
2404
much lower quantities than total opioid material, and it eluted in two broad peaks. The first had a molecular weight of about 13,000 and the second of about 3000. The Met-enkephalin-Arg-Phe immunoreactivity also eluted as two peaks. The first peak eluted in the exclusion fractions and the second eluted at a molecular weight of about 3200. Similar gel filtration chromatography results were obtained for Con A-stimulated supernatants from D1.5, D10.G4.1, and C4 cell lines (data not shown). Gel filtration of rat brain extracts (Fig. 2B) showed that most opioid sequences occur as low-molecularweight (
