Journal of Neuroscience Research 18:82-87 (1987)

Preproenkephalin mRNA in T-Cells, Macrophages, and Mast Cells J. Martin, M.B. Prystowsky, and R.H. Angeletti Divisions of Neuropathology and Immunology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia

The presence of preproenkephalin mRNA in tumor cell lines derived from myeloid and mast cells was analyzed by RNA dot blot hybridization. A B-cell lymphoma was negative for preproenkephalin mRNA, but several T-cell lymphomas were positive. A masto- cytoma and two macrophage cell lines were found to have high levels of preproenkephalin mRNA. Purified natural macrophages and mast cells also possessed easily detectable levels of this mRNA.

Key words: preproenkephalin, opiate peptides, mast cell, macrophages

INTRODUCTION Neuropeptides can influence immune tissue by sev-

eral routes. Studies analyzing the innervation of lym- phoid organs have demonstrated that there are peptides present in neurites and nerve endings, suggesting local effects [Bulloch, 1985; Felten et al, 19851. Alternatively distal effects exerted via the circulation may be mediated by peptides secreted by the pituitary-adrenal axis. Other neuropeptides may reside within nonlymphoid endocrine cells as well, such as thymic epithelial cells [Hall et al, 19851. A final possibility is that the lymphoid cells them- selves may produce neuropeptides. Immunoreactivity for peptides such as endorphins, enkephalins, and adrenocor- ticotropin has been detected [Smith et al, 19861. The mRNA for proopiomelanocortin has now been detected in spleen macrophages [Lolait et al, 19861. The strongest evidence for the endogenous production of peptides in lymphoid cells in a functionally significant manner is the observation that activation of T-helper cells by concana- valin A induces the production of preproenkephalin mRNA to as much 0.5% of total cellular mRNA [Zu- rawski et al, 19861. More recently, Westley and cowork- ers [I9861 have shown that viral infection can induce proopiomelanocortin in spleen cells. Kwon et a1 [1987] have also shown that a T-helper line produces proopio- melanocortin mRNA after lectin stimulation.

Our interest in neuroendocrine-immune interac- tions was stimulated by our observation that chromo- 0 1987 Alan R; Liss, Inc.

granin, a marker protein for neuroendocrine cells, is present in cells within immune tissues [Angeletti and Hickey, 1985, 19871. In order to pursue these observa- tions at the molecular level, preproenkephalin cDNA probes were selected as controls. Preproenkephalin is the 263 amino acid opiate peptide precursor, which can give rise to one leu-enkephalin and six met-enkephalin se- quences by proteolytic cleavages [Comb et al, 1982; Gubler et a1 1982; Noda et al, 1982; Udenfriend and Kilpatrick, 19831. These enkephalin precursors and their receptors may be present in lymphoid tissues. Further- more, opiate peptides have been reported to produce potent effects in immune tissues [Johnson et al, 19821. In order to clearly assess the presence of preproenkephalin in specific cell types of immune tissues, tumor cell lines representing a spectrum of immune cells were selected for study. Analysis of their mRNA by hybridization tech- niques revealed that the mRNA for preproenkephalin is present in lymphoid cell lines and in macrophage and mast cell lines, as well as their natural homologs.

EXPERIMENTAL PROCEDURES Cell Lines

Cloned tumor cell lines were obtained from the American Type Culture Collection. The Thy 1+ S49 lymphomas (TIB 28-36) were derived from a Balb/c mouse tumor [Harris, 1970; Baxter et al, 1971; Ralph, 1973; Ralph et al, 19731. The P815 mastocytoma (TIB 64) was isolated from a DBA/2 mouse [Lundak and Raidt, 1973; Plaut et al, 19731. The J774A. 1 macrophage (TIB 67) is a Balbk mouse line [Ralph and Nakoinz, 1975; Ralph et al, 19751. The WEHI-3 myelomonocytic leukemia, macrophagelike cell line (TIB 68) was isolated from a Balb/c mouse at the Walter and Eliza Hall Institute [Ralph and Nakoinz, 19771. The ELA.IL-2 thymoma (TIB 181) is a mouse cell line [Farrar et al, 19801. The

Received March 31, 1987; revised April 28, 1987; accepted April 29, 1987.

Address reprint requests to Dr. R.H. Angeletti, Neuropathology, University of Pennsylvania School of Medicine, 416 Johnson Pavil- ion, Philadelphia, PA 19104-6079.

PPE mRNA in Lymphoreticular Cells 83

lowed to proceed for an additional 18 hours at 42°C. Blots were washed with 0.1X SSC containing 0.1 % sar- cosyl, first at 20", followed by a final 30-minute wash at 50" [Yoshikawa et al, 19841. Under these conditions of high stringency, no other mRNAs, such as that for pre- proopiomelanocorticotropin, can be detected. Autoradio- graphic detection was performed using Kodak XAR film and DuPont Cronex enhancing screens at -60" for the times noted in figure legends. All chemicals were ob- tained from Aldrich Chemical Co. or Sigma Chemi- cal c o .

A20 B-cell lymphoma (TIB 208) was isolated from a Balb/c mouse [Kim et a1 19791. All cell lines retained morphological, enzymatic, or immunological markers characteristic of the nontransformed cell types. The cells were cultured according to instructions provided by the American Type Culture Collection, using media from Gibco Biologicals, serum from KC Biologicals, and tis- sue culture flasks from Corning.

Cell Isolation Natural peritoneal mast cells were isolated from

peritoneal lavage of Long-Evans rats and purified by a Percoll gradient [Alm, 19831. Percoll was obtained from Pharmacia Fine Chemicals. Mast cells were greater than 95% pure as judged by Wright's stain of cytocentrifuge preparations. Bone marrow cells from tibias and femurs of Balb/c mice were cultured in the presence of L929 cell supernatants as a source of CSF-1, a colony stimu- lating factor specific for macrophages [Stanley, 19861. The adherant cells, which were greater than 95% pure by Wright's stain procedures, were harvested after one week in culture.

mRNA and Plasmid Preparations, Hybridization Total cellular RNA was isolated fom cell lines by

extraction with guanidinium thiocyanate [Chirgwin et al, 19791. Total RNA, not poly A +-selected RNA, was used for membrane hybridizations because the polyadenyla- tion status of preproenkephalin mRNA has not been de- termined [Yoshikawa et al, 19841. The pYSEAl and pYSEC1 plasmids were a generous gift of Steven L. Sabol at the National Institutes of Health [Yoshikawa et al, 19841. These plasmids contain the full length rat preproenkephalin coding region inserted into the SP6 vector [Melton et al, 19841. The pYSEAl plasmid was linearized with SST I (Bethesda Research Laboratories) to provide a template for RNA synthesis using the kit provided by Promega Biotec. The alpha-32P labeled CTP [NEG-OOSX, 22.2 TBq/mmol] was obtained from New England Nuclear Corporation. The antisense probe for binding to mRNA was then purified free of substrate by eluting from a NENSORB-20 column (New England Nuclear Corp.). The pYSEC1 probe, which produces a sense control strand, was similarly labeled after cutting with Sma I (Bethesda Research Laboratories). Total RNA was serially diluted, lyophilized, and denatured before application to Genescreen nylon membrane (New En- gland Nuclear Corp.) as described by Yoshikawa et a1 [ 19841. The membranes were prehybridized overnight at 42°C in the presence of 50% formamide, 1OX Den- hardt's solution, 0.05 M Tris-HC1 pH 7.5, 1 M NaCl, 3.75 mM sodium pyrophosphate, 1 % sarcosyl, 10% dex- tran sulfate, and 100 pglml salmon sperm DNA. The labeled probes were added, and hybridization was al-

RESULTS Hybridization Analysis of Cell Lines

Tumor cell lines were selected from the American Type Culture Collection to represent a spectrum of lym- phoid and accessory cell types. Table I shows a summary of the dot blot hybridization analyses. The B-cell lym- phoma (TIB 208) is a nongranulated cell line exhibiting the following B-cell surface markers: IgG + , Ia + , Fc receptor+. The dot blot for this line was negative at levels of RNA as high as 5 pg. A family of T-lymphoma lines was also tested (TIB 28-36), and showed varying results. Clones of the original lymphoma line have been selected for resistance to different compounds, or surface marker characteristics, as indicated in Experimental Pro- cedures and Table I. The parent line (TIB 28) and the two lines resistant to dexamethasone (TIB33) and thymi- dine (TIB 30) expressed easily detectable levels of the preproenkephalin mRNA as judged by dot blot analysis. In contrast, TIB 36, which does not express surface Thy- 1, did not contain detectable levels of preproenkephalin mRNA. The TIB 181 thymoma cell line, which secretes interleukin 2 and is unrelated to TIB 36, also expressed this mRNA (Table I, Fig. 1).

The most strilung positive results were obtained with nonlymphocyte tumor cell lines. As summarized in Table I and illustrated in Figure 1, the cell lines having the characteristics of mast cells (TIB 64) or macrophages (TIB 67 and 68) exhibited very high levels of preproen- kephalin mRNA, between 100-1,OOO times the amount detected in the T-cell lines. Since the existence of pre- proenkephalin mRNA in the mastocytoma and the mac- rophagelike cell lines was unexpected, we questioned whether its presence might be due to the transformed nature of the cells. Mast cells were purified from perito- neal lavage of Long-Evans rats by use of a Percoll gra- dient [Alm, 19831. Macrophages were prepared from extruded bone marrow by culturing for seven days in the presence of colony stimulating factor 1 (CSF-1) [Stanley, 19861. As seen in Figure 2, these natural cells also contained high levels of preproenkephalin RNA.

84 Martin, Prystowsky, and Angeletti

TABLE I. Presence of Preproenkephalin (PPE) mFWA in Murine Cell Lines

Cell line Properties PPE mRNA

TIB 20ga B cell lymphoma -

TIB 29b-e S49.1 lymphoma (parent) + TIB 30b-e S49. ITB .2 lymphoma, thymidine + TIB 33h-e S49.ITB.4DEX R.63 lymphoma + TIB 36'-" S49 (Thy I-) lymphoma - TIB 181' EL4.IL-2 thymoma (secretes + TIB 64gxh P8 15 mastocytoma + TIB 67I.J J774A. 1 macrophage (secretes + TIB 68k-" WEHI-3 myelomonocytic leukemia, +

and bromodeoxyuridine resistant

(dexamethasone resistant)

interleukin 2)

interleukin 1 and lysozyme)

macrophagelike (secretes lysozyme and interleukin 3)

"Kim et a1 [ 19791. baxter et al [1971]. 'Harris [ 19701. dRalph [1973]. eRalph et a1 [1973]. 'Farrar et al [ 19801. gLundak and Raidt 119731. hPlaut et al [1973]. 'Ralph and Nakoinz [ 19731. 'Ralph et a1 [ 19751. kRalph and Nakoinz [ 19771. 'Farrar et a1 [ 19801. "'Razin et al [ 19841.

DISCUSSION The opiate peptide family, both enkephalins and

endorphins, has been implicated to play functionally sig- nificant roles in the immune system. In vitro assays have shown that one or more of these peptides can increase lymphocyte proliferation and natural killer activity [Gil- man et al, 1982; Johnson et al, 1982; Mathews et al, 19831. At physiological concentrations, met-enkephalin increases antibody dependent cytotoxicity in macro- phages [Foris et al, 19841. At much higher concentra- tions, opiate peptides can affect histamine release from peritoneal mast cells [Grosman et al, 19821. Correspond- ing receptors have been found on lymphocytes but not platelets [Lopker et al, 1980; Mehrishi and Mills, 1983; Reches et al, 1980; Wybran et al, 19791. An endorphin- specific, nonopiate receptor has also been detected on lymphocyte membranes [Hazum et al, 1979; McCain et al, 19821. Although the active opiate peptides might be derived from nerve endings within immune tissues, or arrive via the circulation, it is also possible that there is in situ peptide production. Using immunochemical tech-

1 2 3 64

67

68

181 Fig. 1. Dot blot hybridization of preproenkephalin cRNA (pYSEA1) with total cellular RNA isolated from cell lines. Total cellular RNA from the cell lines indicated were dotted onto Genescreen paper at the following amounts: 1 pg (1); 0.1 pg (2 ) ; and 0.01 pg (3). For the TTB 67 cell line only, the amounts were instead: 0.1 pg (1); 0.01 pg (2); and 0.001 pg (3). The filters were prehybridized and hybridized with an SP6 probe, pYSEA1, containing the coding region for rat pre- proenkephalin. The alpha-32P CTP labeled antisense strand binds to mRNA. The autoradiographic exposure time was 24 hours at -70°C with an enhancing screen.

1 2 3 A

B

Fig. 2. Dot blot hybridization of preproenkephalin cRNA with total cellular RNA isolated from cell lines. Total cellular RNA isolated from purified rat peritoneal mast cells and mouse bone marrow macrophages were dotted onto Genescreen membrane at the following amounts: 0.1 pg (1); 0.01 pg (2); and 0.001 pg (3). Row A: mast cell RNA; Row B: macro- phage RNA. The autoradiographic exposure time was 22 hours

niques, immunoreactive adrenocorticotropin and endor- at - 70°C with an enhancing screen.

PPE mRNA in Lymphoreticular Cells 85

detection of preproenkephalin mRNA in primary mast cells and macrophages was of particular importance. The first question to be answered is whether this mRNA is translated into the effective gene product. However, im- portant issues of biological relevance must also be ad- dressed. Does the presence of preproenkephalin have any relationship to the phagocytic function of macrophages? Are the enkephalin products secreted via stimuli that cause either cell type to secrete? Does the presence of preproenkephalin mRNA reflect the functional status of the cell? For example, the bone marrow macrophages prepared in culture are Ia- , and thus have no antigen- presenting capability. We must examine whether a change to Ia+ positivity alters the level of preproenkephalin mRNA. The potential for Ia expression is reduced in the neonate [Lu and Unanue, 19821. Although all macro- phages may be derived from a common Ia negative stem cell [Scher et al, 19821, Ia expression is known to vary depending on the tissue of residence, such as spleen, thymus, and peritoneum [Lu et al, 19811. Ia expression is induced by immunologic stimuli [Beller et al, 19801 and can be transient [Beller and Unanue, 19811. Macro- phages also have cytocidal functions and secrete interleu- kin 1. They can be stimulated by several growth factors. Thus, a further exploration of the effect of experimental manipulation on preproenkephalin mRNA levels and the relevance to macrophage function should be pursued.

Mast cells are found in many locations: lung, intes- tine, peritoneum, blood, skin, and other tissues. They are involved in immediate hypersensitivity reactions, bind IgE, and secrete histamine, proteoglycans, and other biologically active substances. Tissue mast cells may also control local blood flow and stimulate proliferation of endothelial cells [Marks et al, 1986; Benyon et al, 19871. Mast cells can be generated from bone marrow by cultur- ing in the presence of interleukin 3 [Razin et al, 19841. Although mast cells may have a common precursor [Ko- bayashi, 19861, there is increasing evidence for their heterogeneity. Specific populations may vary in the type of proteoglycans and proteases produced and may have altered sensitivities to different nonimmunologic secre-

phins have been detected in leukocytes [Lolait et al, 1984; Smith et al, 19861. However, it has been suggested that some of this activity may be associated with the interferon-alpha molecule [Blalock and Smith, 1981 ; Smith and Blalock, 19811. However, recent evidence confirms that the proopiomelanocortin mRNA is ex- pressed in macrophages [Lolait et al, 19861. Strong evi- dence for the synthesis of opiate peptides by endogenous immune cells was obtained by Zurawski and coworkers [1986]. These investigators cloned the cDNA for an mRNA species found abundantly in lectin-activated T- helper cells, but undetectable in unstimulated cells. The sequence of this cDNA was that of the previously identi- fied preproenkephalin. More recently, a cDNA for pro- opiomelanocortin has been cloned from a lectin-activated T helper lymphocyte [Kwon et al, 19871. These investi- gators also found that this mRNA is produced in the T- helper cells after stimulation with concanavalin A, but not with the growth factor interleukin 2. Thus, it is possible that the force of biologically active peptides in immune tissues may be diverse and may be either long- or short-range, transient or persistent in nature. In some cases, peptide production may be dependent on the func- tional state of activation of the lymphoid cells.

In the present investigation, we examined tumor cell lines for the presence of preproenkephalin mRNA using a specific antisense RNA probe. A B-cell lym- phoma possessing the classical surface marker phenotype of normal B cells did not possess any detectable pre- proenkephalin mRNA. The TIB 181 thymoma, which secretes interleukin 2, was strongly positive. A family of T-cell lymphomas was also examined. Some of the mem- bers of this family were positive when tested with the preproenkephalin probe. However, there was no appar- ent correlation with phenotype of drug resistance. The cell line that was negative for preproenkephalin mRNA was also the only one that was negative for the Thy-1 surface marker. Although this observation is intriguing, the sample number is too small to assess whether there is a significant correlation. More T cell lines will need to be examined. In neuroendocrine systems, preproenke- phalin is stored within and secreted from- specialized I tory stimuli, such as substance P [Barrett et al, 1986; vesicles. The lymphomas tested in this study are not Benyon et al, 1987; Lee et al, 1985; Razin et al, 1984; known to possess secretory vesicles. Thus, important Stevens et al, 19861. The detection of preproenkephalin questions are whether these cells possess translated pre- mRNA in mastocytomas and peritoneal mast cells raises proenkephalin as well as the mRNA and whether they the question of whether its presence correlates with secrete this product by a constitutive instead of a vesicu- known differences in mast cell products. It also suggests lar mechanism. possible roles that opiate peptides might play in hyper-

The observation that mast cell lines and macro- sensitivity and in local effects on surrounding tissues. phage cell lines were positive when tested with the The investigation of the role of preproenkephalin in pYSEA1 probe raised the issue that our results reflected macrophage and mast cell function should now be ex- the transformed phenotype of these cell lines, and not a plored using both hybridization and immunochemical property of potential biological significance. Thus, the techniques.

86 Martin, Prystowsky, and Angeletti

ACKNOWLEDGMENTS This work was supported by grants from the Na-

tional Institutes of Health: NS-22697 (RHA) and AI- 21681 (MBP). MBP is a Hartfor Foundation Fellow.

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