Related Experiment Videos
In vitro cytotoxicity of peritoneal macrophages activated with Mycobacterium smegmatis
Abstract:
Incorporation of 3H-TdR into EL4 leukemic cells in vitro was inhibited by peritoneal exudate cells (PEC) harvested from syngeneic C57BL/6J mice given an intraperitoneal (i.p.) injection of 1x10(7) viable Mycobacterium smegmatis ATCC 607 (Smeg) 4 days before. This phenomenon was also observed in the following five systems of PEC from animals and syngeneic tumor cells: C57BL/6J mice and B16 melanoma; DBA/2 mice and P815 mastocytoma; SWM/Ms mice and K5 fibrosarcoma; BALB/c, nu/nu mice and KKN-1 fibrosarcoma; and strain 2 guinea pigs and line-10 hepatoma. The in vitro cytotoxicity of the PEC activated by viable Smeg was much higher than those activated by dead-Smeg, viable BCG or proteose peptone. The activity of the adherent fraction of the PEC was stronger than that of the nonadherent one, and not influenced by either anti-theta or anti-mouse lymphocyte rabbit sera. The PEC induced with Smeg 4 days before contained a large population of mononuclear cells (88.9%) and a significant level of polymorphonuclear cells (PMN) (3.2%), and showed a much higher cytotoxicity than the PEC induced with Smeg 3 hr before, which contained a much larger population of PMN (71.9%), suggesting that PMN were not the effector cells in this system. In vitro and in vivo treatment with macrophage-inhibitors such as carrageenan, trypan blue and cytochalacin B, reduced the activity of the PEC. All of these facts suggested macrophages as the effector. Viable macrophages were required for the growth inhibition of EL4 in vitro: gamma-ray irradiated or freeze-thawed macrophages were ineffective. Kinetic studies revealed that inhibition of 3H-TdR incorporation into EL4 cells started within 3 hr of incubation together with the activated macrophages at an effector to target (E/T) ratio of 5, and the incorporation decreased gradually with the lapse of incubation time. On the other hand, 51Cr release from labelled EL4 was undetected when the E/T ratio was 5 but detected at on E/T of 10 or more. Even at the higher E/T ratio, at least 10 hr were needed until the release of 51Cr, suggesting that the activated macrophages produced growth inhibition of tumor cells followed by cell destruction.
Insights
Mycobacterium smegmatis activates peritoneal exudate cells (PECs) to inhibit tumor cell growth. Activated macrophages, not polymorphonuclear cells, are the effector cells, causing tumor cell growth inhibition and subsequent destruction.
Area of Science:
- Immunology
- Cancer Research
- Microbiology
Background:
- Peritoneal exudate cells (PECs) are immune cells found in the peritoneal cavity.
- Tumor cells, such as EL4 leukemic cells, are often used in cancer research to study tumor growth and potential treatments.
Purpose of the Study:
- To investigate the role of Mycobacterium smegmatis (Smeg) in activating peritoneal exudate cells (PECs) for anti-tumor activity.
- To identify the specific immune cells responsible for the anti-tumor effects induced by Smeg activation.
Main Methods:
- In vitro incubation of EL4 leukemic cells with PECs from syngeneic mice injected with viable Mycobacterium smegmatis.
- Assessing tumor cell proliferation using 3H-TdR incorporation and cell lysis using 51Cr release assays.
- Utilizing macrophage inhibitors and analyzing PEC composition (mononuclear cells vs. polymorphonuclear cells).
Main Results:
- Viable Smeg significantly enhanced PEC cytotoxicity against EL4 leukemic cells and other tumor cell lines.
- Activated macrophages, identified as mononuclear cells, were the primary effector cells, not polymorphonuclear cells.
- Macrophage inhibitors reduced PEC activity, and non-viable or inactivated macrophages were ineffective.
Conclusions:
- Mycobacterium smegmatis activates macrophages within PECs, leading to potent anti-tumor activity.
- Activated macrophages inhibit tumor cell growth and subsequently cause cell destruction.
- This study highlights the potential of Smeg-activated macrophages as a therapeutic strategy against tumors.