Zbl. Bakt. 277, 357-363 (1992) © Gustav Fischer Verlag, StuttgartlNew York
Thymocyte Proliferation and Maturation in Response to Staphylococcal Lipoteichoic Acid Y. OHSHIMAl, H. L. K02, and G. PULVERER 2
J.
BEUTH h
,
H. BURRICHTER 3 ,
Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 213, Japan 2 Institute of Medical Microbiology and Hygiene, University of Cologne, 5000 Cologne 41, Germany 3 Institute of Clinical Chemistry, University of Cologne, 5000 Cologne 41, Germany 1
Received May 18, 1992 . Accepted May 29, 1992
Summary Lipoteichoic acid (LT A) from Staphylococcus saprophyticus strain S1 could be shown to induce thymocyte proliferation and maturation in BALB/c-mice after systemic administration. The increase in thymocyte numbers per mg organ weight was statistically significant. Determination of thymic lymphatic subsets revealed a considerable up-regulation of mature cells expressing helper/inducer (L3T4) or cytotoxic/suppressor (Lyt-2) phenotypes. Thus administration of staphylococcal LT A obviously accelerated murine thymocyte proliferation and maturation. Counts of BALB/c-mouse peripheral blood lymphocytes (PBL) revealed no evident fluctuation within one week after LT A administration, however, statistically significant increases could be detected two weeks after treatment. The determination of activated PBL expressing Il-2 receptors suggested that injection of staphylococcal LTA apparently induced an immunostimulation since those cells were significantly enhanced within one week after LT A administration.
Zusammenfassung Die systemische Gabe von Lipoteichonsaure (LT A), extrahiert aus Staphylococcus saprophyticus Stamm Sl bewirkte eine beschleunigte Proliferation und Ausreifung von Thymozyten in BALBIc-Mausen. Die Steigerung der Thymozytenzahl pro mg Organgewicht war statistisch signifikant. Die Analyse lymphatischer Subpopulationen des Thymus liefs eine Steigerung reifer Zellen mit Helfer (L3T4) bzw. Suppressor (Lyt-2)-Phanotyp erkennen. Die Zahl peripherer BALBIc-Maus-Lymphozyten (PBL) unterschied sich eine Woche nach LTA-
'f
Corresponding author
358
Y. Ohshima, H. L. Ko,
J. Beuth, H. Burrichter,
and G. Pulverer
Injektion nicht wesentlich von der Lymphozytenzahl unbehandelter Kontrolltiere, zwei Wochen nach der LTA-Gabe waren allerdings statistisch signifikant erh6hte PBL-Zahlen nachweisbar. Die Analyse des Aktivierungszustandes der PBL ergab eine signifikant vermehrte Interleukin (11)-2 Rezeptorexpression, die ein Korrelat der immunaktiven Wirkung von LTA reprasentiert.
Introduction The ability of numerous bacteria and bacterial products to modulate the immune response to unrelated antigens is well documented (9,18). Lipoteichoic acid (LTA), an amphipathic molecule originating from the cell walls of most gram-positive bacteria, has been shown to bind to specific receptors on mammalian cell membranes, to mediate the attachment of certain gram-positive bacteria to host tissue and to induce a variety of immune responses (2, 3,10, 14, 15,22). Intrathymic T-cell differentiation is a process in which immature thymocytes expand and develop by undergoing complicated maturational events leading to the acquisition of immunocompetence and subsequent migration to the periphery (5, 19). The objective of this study was to evaluate the influence of staphylococcal LT A on thymocyte proliferation, maturation and emigration into peripheral blood.
Materials and Methods Animals. BALB/c-mice (Charles River Wiga Breeding Co., Sulzfeld, Germany), 6-8 weeks old, weighing about 16-18 g were used for these studies. The animals were kept in plastic cages and allowed free access to food and water. Extraction of lipoteichoic acid (LTA). LTA was extracted by the method of Moskowitz (13) with minor modifications as described elsewhere (15, 16). Briefly, S. saprophyticus (strain S 1) was harvested by centrifugation, washed with phosphate buffered saline (PBS; Flow Laboratories GmbH, Meckenheim, Germany) and extracted with aqueous phenol (46% v/v). The aqueous phase containing the LTA was removed, treated with chloroforme: methanol (2: 1 v/v) overnight at 4°C, dialyzed against distilled water and lyophilized. The crude extract was loaded on a Sepharose CL-B column (1.5 by 80 cm; Pharmacia Fine Chemicals, Freiburg, Germany,) and eluted with 0.2 M ammonium acetate. Fractions within each of the phosphorus containing peaks were dialyzed against distilled water and lyophilized. Quantitative and chemical analyses were performed as previously described (14) and finally, LTA wa, determined by the inhibition of heat coagulation of human albumin according to Teti et al. (22). Experimental desty,n. Groups of 5 BALBIc-mice were intraperitoneally (i.p.) injected with staphylococcal LTA iO. L 0.25, 0.5 mg per injection, dissolved in 0.2 ml PBS, which proved to be optiinal in preliminary experiments) for 2 subsequent days. Control mice were equally treated with PBS. 7 and 14 days after terminating the LT A treatment mice were sacrificed, their thymuses remo\'ed and cells were separated and counted as described (4, 11). Parallely, peripheral blood wa, drawn for analyses of white blood cell count and differentiation in accordance with 'it
Thymocyte Proliferation and Maturation in Response to Staphylococcal Lipoteichoic Acid Y. OHSHIMAl, H. L. K02, and G. PULVERER 2
J.
BEUTH h
,
H. BURRICHTER 3 ,
Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 213, Japan 2 Institute of Medical Microbiology and Hygiene, University of Cologne, 5000 Cologne 41, Germany 3 Institute of Clinical Chemistry, University of Cologne, 5000 Cologne 41, Germany 1
Received May 18, 1992 . Accepted May 29, 1992
Summary Lipoteichoic acid (LT A) from Staphylococcus saprophyticus strain S1 could be shown to induce thymocyte proliferation and maturation in BALB/c-mice after systemic administration. The increase in thymocyte numbers per mg organ weight was statistically significant. Determination of thymic lymphatic subsets revealed a considerable up-regulation of mature cells expressing helper/inducer (L3T4) or cytotoxic/suppressor (Lyt-2) phenotypes. Thus administration of staphylococcal LT A obviously accelerated murine thymocyte proliferation and maturation. Counts of BALB/c-mouse peripheral blood lymphocytes (PBL) revealed no evident fluctuation within one week after LT A administration, however, statistically significant increases could be detected two weeks after treatment. The determination of activated PBL expressing Il-2 receptors suggested that injection of staphylococcal LTA apparently induced an immunostimulation since those cells were significantly enhanced within one week after LT A administration.
Zusammenfassung Die systemische Gabe von Lipoteichonsaure (LT A), extrahiert aus Staphylococcus saprophyticus Stamm Sl bewirkte eine beschleunigte Proliferation und Ausreifung von Thymozyten in BALBIc-Mausen. Die Steigerung der Thymozytenzahl pro mg Organgewicht war statistisch signifikant. Die Analyse lymphatischer Subpopulationen des Thymus liefs eine Steigerung reifer Zellen mit Helfer (L3T4) bzw. Suppressor (Lyt-2)-Phanotyp erkennen. Die Zahl peripherer BALBIc-Maus-Lymphozyten (PBL) unterschied sich eine Woche nach LTA-
'f
Corresponding author
358
Y. Ohshima, H. L. Ko,
J. Beuth, H. Burrichter,
and G. Pulverer
Injektion nicht wesentlich von der Lymphozytenzahl unbehandelter Kontrolltiere, zwei Wochen nach der LTA-Gabe waren allerdings statistisch signifikant erh6hte PBL-Zahlen nachweisbar. Die Analyse des Aktivierungszustandes der PBL ergab eine signifikant vermehrte Interleukin (11)-2 Rezeptorexpression, die ein Korrelat der immunaktiven Wirkung von LTA reprasentiert.
Introduction The ability of numerous bacteria and bacterial products to modulate the immune response to unrelated antigens is well documented (9,18). Lipoteichoic acid (LTA), an amphipathic molecule originating from the cell walls of most gram-positive bacteria, has been shown to bind to specific receptors on mammalian cell membranes, to mediate the attachment of certain gram-positive bacteria to host tissue and to induce a variety of immune responses (2, 3,10, 14, 15,22). Intrathymic T-cell differentiation is a process in which immature thymocytes expand and develop by undergoing complicated maturational events leading to the acquisition of immunocompetence and subsequent migration to the periphery (5, 19). The objective of this study was to evaluate the influence of staphylococcal LT A on thymocyte proliferation, maturation and emigration into peripheral blood.
Materials and Methods Animals. BALB/c-mice (Charles River Wiga Breeding Co., Sulzfeld, Germany), 6-8 weeks old, weighing about 16-18 g were used for these studies. The animals were kept in plastic cages and allowed free access to food and water. Extraction of lipoteichoic acid (LTA). LTA was extracted by the method of Moskowitz (13) with minor modifications as described elsewhere (15, 16). Briefly, S. saprophyticus (strain S 1) was harvested by centrifugation, washed with phosphate buffered saline (PBS; Flow Laboratories GmbH, Meckenheim, Germany) and extracted with aqueous phenol (46% v/v). The aqueous phase containing the LTA was removed, treated with chloroforme: methanol (2: 1 v/v) overnight at 4°C, dialyzed against distilled water and lyophilized. The crude extract was loaded on a Sepharose CL-B column (1.5 by 80 cm; Pharmacia Fine Chemicals, Freiburg, Germany,) and eluted with 0.2 M ammonium acetate. Fractions within each of the phosphorus containing peaks were dialyzed against distilled water and lyophilized. Quantitative and chemical analyses were performed as previously described (14) and finally, LTA wa, determined by the inhibition of heat coagulation of human albumin according to Teti et al. (22). Experimental desty,n. Groups of 5 BALBIc-mice were intraperitoneally (i.p.) injected with staphylococcal LTA iO. L 0.25, 0.5 mg per injection, dissolved in 0.2 ml PBS, which proved to be optiinal in preliminary experiments) for 2 subsequent days. Control mice were equally treated with PBS. 7 and 14 days after terminating the LT A treatment mice were sacrificed, their thymuses remo\'ed and cells were separated and counted as described (4, 11). Parallely, peripheral blood wa, drawn for analyses of white blood cell count and differentiation in accordance with 'it
