Proc. Nati. Acad. Sd. USA Vol. 75, No. 7, pp. 34003404, July 1978 Immunology Tuftsin (an Ig-associated tetrapeptide) triggers the immunogenic function of macrophages: Implications for activation of programmed cells (tuftsin receptor/peptide hormones) E. TZEHOVAL*, S. SEGAL*, Y. STABINSKYt, M. FRIDKINt, Z. SPIRER*, AND M. FELDMAN* Departments of * Cell Biology and tOrganic Chemistry, The Weizmann Institute of Science, Rehovot, Israel; and * Pediatrics Department A, Municipal Covernmental Medical Center, Tel Aviv, Israel Communicated by Abraham White, April 24,1978 ABSTRACT The immunoglobulin heavy-chain-associated bradykinin (11) were synthesized as described. The "anti-al- tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), known for its phago- lergic peptide," Asp-Ser-Asp-Pro-Arg, (12) was also synthesized cytosis-stimulating activity, was found to augment the anti- gen-specific, macrophage-dependent education of T lympho- in our laboratory (unpublished). Luteinizing hormone-releasing cytes.The investigation of stereospecific characteristics of the hormone (luliberin) was kindly donated by Ayerst Research tetrapeptide, by use of structural analogs with different modi- Laboratories, Montreal, Canada. Keyhole limpet hemocyanin fications, revealed strict structural requirements for eliciting (KLH) was purchased from Calbiochem (Venice, CA; lot 53006, the immunogenic activity of macrophages. It was found that A grade). 2-Mercaptoethanol was ordered from Eastman Kodak the most important moiety for its activity is the dipeptide Pro- Co., Rochester, NY. Dulbecco's modified Eagle's medium and Arg. Ths finding is of interest in view of the appearance of this Dulbecco's phosphate-buffered saline, pH 7.4, (Pi/NaCI) were particular dipeptide in other bioregulatory peptides, including supplied by the Cell Biology Department. RPMI 1640 (catalog many of the peptide hormones. The significance of the ap- pearance of a commoil structure in such molecules, which may no. H-18) was purchased from GIBCO (Grand Island, NY). All act through specific receptors on different target cells, is dis- culture media were supplemented with penicillin (100 units/ cussed. ml), streptomycin (100 ,g/ml), and sodium bicarbonate (24 mM). Numerous studies have demonstrated the central role of T cells Preparation of Macrophages. BALB/c mice were injected in humoral and cellular immune responses. In addition to intraperitoneally with 3 ml of thioglycollate broth (Difoo). On functioning as effector cells, T cells may also augment as helpers the 5th day, the mice were killed by suffocation, with dry ice, or suppress the function of other cellular components of the and injected intraperitoneally with 10 ml of Pi/NaCl containing immune system (1). The activation by antigen of T cells, re- 5 units of heparin per ml (Thromboliquin N.V., Organon, Oss, sulting in antibody production, seems to depend on the presence Holland). The peritoneum was gently massaged and the peri- of macrophages (2-5). The presentation of antigen via the toneal fluid was aspirated with a 10-ml syringe. The cell sus- macrophage determines the triggering and generation of an pension was centrifuged in a refrigerated centrifuge at 200 X active T cell (4, 6). Yet, the mechanism that controls the acti- g for 10 min. The supernatant was discarded and the cells were vation of the immunogenic potential of the macrophages resuspended in Dulbecco's modified Eagle's medium, adjusted themselves is largely unknown. to a concentration of 2 X 106 peritoneal cells per ml. The sus- Studies of Najjar and his coworkers (7, 8) and those carried pensions contained approximately 90% macrophages and 10% out in our laboratory (9, 10)showed that the basic tetrapeptide lymphoid cells. L-threonyl-L-lysyl-L-prolyl-L-arginine, tuftsin, originating from In Vitro "Education" of Spleen Cells. In order to educate a specific leukophilic gamma globulin fraction, exhibits a high splenic lymphocytes, we used a method developed in our lab- activation potential for phagocytic cells: polymorphonuclear oratory and described elsewhere (13). In brief, 10 ml of mac- leukocytes (PMN) and macrophages. These experiments rophage suspension was plated on a 100-mm plastic tissue cul- demonstrated that tuftsin increases the phagocytic activity of ture petri dish (Falcon no. 3003, Falcon Plastics, Oxnard, CA). these cells (7-10) and potentiates several of their enzymatic The petri dishes were incubated overnight at 370 in a humi- systems involved in the pentose shunt (as assessed by en- dified atmosphere of 5% CO2 in air, to provide maximum ad- hancement of the nitroblue tetrazolium reduction test) (9, 10). herence of macrophages as a monolayer. The supernatant was led us to then discarded and the cultures were washed three times with These observations investigate possible stimulatory Pi/NaCl to remove most of the lymphocytes and to obtain as activity of tuftsin on the macrophage-dependent immune pure a population of macrophages as possible. To feed the "education" of T cells. The results suggest a general principle macrophages with antigen, we added 4 ml of Dulbecco's associated with the activating signal for certain classes of modified Eagle's medium containing 50 gg of KLH per ml to functionally programmed cells. each petri dish for 4 hr at 37°. In the experimental groups the MATERIALS AND METHODS medium also contained tuftsin or its analogs at various con- Mice. We used BALB/c female mice, 6-8 weeks old, sup- centrations. At the end of this incubation, excess free antigen plied by the Animal Breeding Center of the Weizmann Insti- was washed out by three sequential washes with an excess of tute. Pi/NaCl. At this stage, 10 ml of spleen cells (107 cells per ml) Chemicals and Media. Tuftsin and its analogs (9, 10, §) and with more than.90% viability (as assessed by the trypan blue dye The costs of publication of this article were defrayed in part by the Abbreviations: PMN, polymorphonuclear leukocytes; KLH, keyhole payment of page charges. This article must therefore be hereby marked limpet hemocyanin; Pi/NaCl, phosphate-buffered saline. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate § Stabinsky, Y., Fridkin, M., Zakuth, V. & Spirer, Z. (1977) Proceedings this fact. of the 44th Meeting, Israel Chemical Society, Abstr. MN-10. 3400 Downloaded by guest on September 29, 2021 Immunology: Tzehoval et al. Proc. Nati. Acad. Sci. USA 75 (1978) 3401 exclusion test) were added on top of the antigen-fed monmolyers and incubated at 370 in an atmosphere of 5% CO2 in air over- night. The nonadherent cells were then collected gently, re- seeded on another set of petri dishes (10 ml per dish), and in- cubated for 90 min to adsorb as many residual adherent cells as possible and thus minimize contamination with macro- phages. The cells were then irradiated with 1000 rads, using a Der- mavolt x-ray machine (Siemens x-ray tube: 56 kV, 20 mA, 0.5 Al filter, at a dose rate of 750 rads/min). The suspension was collected, centrifuged, and washed twice at 250 X g for 10 min. The supernatants were discarded and the cells were resus- pended in Eagle's medium and adjusted to a concentration of -400 108 nucleated cells per ml. Fifty microliters of this suspension were injected in the hind foot pads of syngeneic mice. On the 7th day, the popliteal lymph nodes were removed and a cell suspension was prepared in Pi/NaCl. The cells were washed 300- once and resuspended in culture medium (RPMI 1640 sup- plemented with 0.5% syngeneic mouse serum and 50 tiM 2- mercaptoethanol) to a concentration of 5 X 106 cells per ml. A 0. 1-ml sample of these cells was cultured in tissue microtiter 200 plates (Greiner, Nurtingen, Germany) in the presence of anti- gen or control reagents at 370 in a humidified atmosphere of 5% CO2 in air. After 72 hr, 2 of tritiated thymidine ,Ci (Israel 10O AEC, Negev, Israel) was added and the cells were allowed to incubate for 4 hr. Cells were then collected by a "Titertek" cell harvester (Skatronas, Lierbyen, Norway) on a glass filter, washed twice with saline, dried, and placed in Bray's scintil- lation fluid. Radioactivity was measured in a Packard Tri-Carb 0 20 160 liquid scintillation spectrometer. The results were expressed [TuftsinJ, nM in percent activation as to not compared controls treated with FIG. 1. Effect oftuftsin on augmentation ofimmunogenic activity tuftsin. ofantigen-fed macrophages. Macrophages were incubated with KLH and tuftsin at the indicated concentrations. Spleen cells were seeded RESULTS on those monolayers, irradiated, and injected in the hind foot pads. The in vitro antigen-specific response of 5 X 105 lymphocytes derived Guided by the fact that tuftsin augments the phagocytic activity from the popliteal lymph nodes to 0.5 Mg of KLH (0) or 0.05 Mg of of PMN and of macrophages, we designed our first experiment KLH (0) was determined by [3Hlthymidine incorporation. Results to test whether the peptide has the ability also to potentiate the are expressed as percent activation, compared to the control group immunogenic activity of macrophages. Macrophages were untreated with tuftsin and incubated with KLH alone. For experi- mental details, see legend to Fig. 2. incubated in the presence of various concentrations of tuftsin with a constant amount of antigen. Spleen cells were seeded as with that of some peptides unrelated to tuftsin. Such exper- for education on the macrophage monolayer and were then iments tested also whether there is a correlation between the injected into the hind foot pads of syngeneic mice. The popliteal tuftsin's capability to augment phagocytosis and its function lymph-node cells were then tested for response to antigen. The as an immunogenic stimulator. results (Fig. 1) indicate that tuftsin did produce a significant, concentration-dependent potentiation of the education capa- Table 1. Relationship of tuftsin-induced macrophage-mediated bility of antigen-fed macrophages.