Peptides 25 (2004) 91–94
Immunohistochemical staining of endomorphin 1
and 2 in the immune cells of the spleen
, D.S. Jessop, M.S. Harbuz
Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK
Received 8 April 2003; accepted 17 November 2003
Endomorphin 1 (EM-1) and EM-2 have been widely reported in the cells of the central nervous system (CNS) but limited research has
been done regarding their distribution in the peripheral system. The occurrence of EM-1 and -2 in the spleen as measured by RIA and their
ability to mediate immune function imply a role for EMs in this area. The current study examines the localization of EM-1 and -2 in the
immune cells of the spleen of male and female rats via an immunohistochemical procedure. In both genders, EM-1 and -2 immunoreactive
staining was predominantly present in macrophages and B cells with minimal EM immunoreactive staining in T cells. This is the ﬁrst
evidence of a differential distribution of EM-1 and -2 in cells of the immune system.
© 2004 Published by Elsevier Inc.
Keywords: Endomorphin; Spleen; Immunohistochemistry; Macrophages; B cells; T cells; Gender
Since the discovery of the tetrapeptides—endomorphin 1
and 2 (EM-1 and -2)—much research has focused on their
distribution and purpose. Their high selectivity and afﬁnity
for the opiate receptor suggests that they are the true
endogenous ligands for these receptors ( for review).
The majority of work has concentrated on the CNS in
identifying the presence of EM-1 and -2 immunoreactivity
in, for example, the hypothalamus and spinal cord .
Such expression is similar to that found for other opioids.
In contrast relatively little exploration of peripheral EM-1
and -2 expression has been undertaken. The occurrence of
neuropeptides, such as ␤-endorphin  and dynorphin 
in immune tissue suggested that EM-1 and -2 may also
be co-expressed here. The demonstration of EM-1 and
-2 immunoreactivity in rat spleen and thymus tissue via
RIA and HPLC techniques  conﬁrmed this proposition.
However, this study did not distinguish where, at a cellular
level, EMs were expressed. The occurrence of receptors
on macrophages in the rat  and in T and B human cell
lines  would suggest that, due to their high afﬁnity for
such receptors, EMs would also be present in these areas.
The demonstration of EM immunocytochemical staining in
immune cells of popliteal lymph nodes  supports this
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E-mail address: email@example.com (J.V. Seale).
proposition. The increase in EM-like immunoreactivity due
to alterations in lymphocyte levels following hind-paw in-
ﬂammation implies a mediating effect of immune function
on EM expression . An interaction between EMs and
the immune system is further supported by the ability of
EM-1 to attenuate the inﬂammatory response to substance
P in a rat model of acute inﬂammation .
The increased susceptibility of females to autoimmune
diseases  and the differential inﬂuence of gonadal hor-
mones on immune activity ( for review) leads to the
question whether such differences might be associated with
sexual dimorphism in EM expression.
The current study sought to determine, via double im-
munohistochemical labelling, the cellular expression of
EM-1 and -2 in rat spleen tissue and whether any sex
differences in EM expression occurs in splenocytes.
Eight adult Lewis (four males and four females) rats
(225–250 g) were housed in cages of four. Rats were kept
on a 12 h light:12 h dark schedule with ad libitum access
to food and water. Rats were decapitated and spleens ﬁxed
in 4% paraformaldehyde in 1× phosphate buffer for 24 h,
0.1 M sodium phosphate buffer twice for 24 h each and
0196-9781/$ – see front matter © 2004 Published by Elsevier Inc.