Evidence for the synthesis and release of strongly immunosuppressive, noncytotoxic substances by Streptococcus intermedius.

Abstract

A B S T R A C T Products secreted by Streptococcus intermedius were studied for their effects on the immune response. Three different preparations of crude extracellular products from S. intermedius (CEP-Si) were found to have powerful suppressor activity in vitro as shown by inhibition of human lymphocyte proliferation (uptake of [3H]thymidine) and protein synthesis in response to a wide variety of stimulants, including mitogens and antigens, and suppression of plaque formation by human cells in response to sheep erythrocytes. CEP-Si was noncytotoxic, because cells incubated with high concentrations of CEP-Si and subsequently washed were viable and recovered their ability to respond to mitogens, and because leukocyte migration was not inhibited by CEP-Si, nor was the release of leukocyte migration inhibitory factor from sensitized lymphocytes. The possibility of antigen or mitogen competition was excluded. The effects of CEP-Si in vitro were time dependent and did not require the presence of monocytes. Cells pretreated with CEP-Si and then washed suppressed plaque formation by fresh autologous cells in highly stimulated cultures. CEP-Si injected into C57BL/6 mice also strongly suppressed their immune response to sheep erythrocytes, and the in vivo suppression was correlated with the effects of CEP-Si in vitro.

Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

“…We compared the behavior ofCEP-Si with other previously described noncytotoxic immunosuppressor substances obtained from microorganisms such as streptococcal extracts (2), streptococcal lipoteichoic acid (3), and bacterial enzymes like L-asparaginase (4), L-glutaminase (5), and ribonuclease (6); we concluded that CEP-Si was different from these substances mainly in the time-course of its effects and in the possible specificity of the resulting immunosuppression in relation to the stimulant. Other differences between the in vivo and in vitro effects of CEP-Si and both lipoteiochoic acid and streptococcal extracts were also demonstrated, and preliminary results on the purification of CEP-Si were reported (1). We have now confirmed our preliminary results, and in this report we describe the biochemical characteristics of the highly purified immuinosuppressive material obtained from CEP-Si.…”

“…S. initerniedius was cultured and CEP-Si was obtained by the proceduires described previously in detail (1,7). 1 Abbreviationis uised in this pap)er: CEP-Si, Streptococcus initerintedius; MLC, mixed lymphocyte cultures; PHA, phytohemagglutinin; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SRBC, sheep erythrocytes; TEMED, N,N,N',N'-tetramethylenediamine.…”

“…J.) and metrizoate by the method of Boyum (8) and cultured, as described previously (1), according to the method of Du Bois et al (9) in minimal essential medium (Gibco Diagnostics Laboratories, Lawrence, Mass.) supplemented with 20% fresh heat-inactivated human AB serum and antibiotics.…”

“…CEP-Si or its subfractions were added at the beginning of the MLC or of unstimulated cultures and at different intervals in the case of PHA-stimulated cultures. The suppressor effects of the most purified subfraction on both PHA-and MLC-stimulated cultures were estimated by means of the mathematical formulas described previously (1). On the basis of these questions, 1 U of PHA or MLC biological activity was considered the amount of suppressor substance producing a 50% reduction in [3H]-thymidine uptake as compared with controls (PHA-or MLCstimulated cultures without CEP-Si or its subfractions) (see Fig.…”

“…We recently reported that crude extracellular products of Streptococcus intermedius (CEP-Si)l contain an extremely powerful noncytotoxic immunosuppressor substance(s) active both in vivo and in vitro (1). We compared the behavior ofCEP-Si with other previously described noncytotoxic immunosuppressor substances obtained from microorganisms such as streptococcal extracts (2), streptococcal lipoteichoic acid (3), and bacterial enzymes like L-asparaginase (4), L-glutaminase (5), and ribonuclease (6); we concluded that CEP-Si was different from these substances mainly in the time-course of its effects and in the possible specificity of the resulting immunosuppression in relation to the stimulant.…”