Macrophage-mediated Tumor Cell Killing Research Articles

Intratumoral MPL Induces Th1 Polarization and Augments In Situ Vaccination Generated by Radiation and Checkpoint Blockade

<h3>Purpose/Objective(s)</h3> Radiation therapy (RT) has been demonstrated to generate an in situ vaccination effect in murine models and in cancer patients, however this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the in situ vaccination regimen consisting of combination RT and ICI. <h3>Materials/Methods</h3> We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Mice bearing B78 or Myc-CaP tumors were randomized to receive RT (12 Gy), RT + anti-CTLA4 (C4; 100 µg IP injection days 3, 6, 9 after RT), MPL (20 µg IT injection days 5, 7, 9 after RT), RT+C4+MPL, or PBS control. To model metastatic disease, 250,000 B16 melanoma cells (parental to B78) were injected via tail vein immediately following RT. To evaluate the effect of MPL on the irradiated tumor microenvironment, a separate cohort of B78 tumor bearing mice from each treatment group were sacrificed 15 days following RT, and tumor together with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling and serum was collected for analysis of anti-tumor antibody populations. <h3>Results</h3> Combination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared to RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment towards M1 macrophage and Th1 CD4 T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1 and M1 associated proinflammatory cytokines. T cells co-cultured with MPL-stimulated macrophages significantly increased expression of the activation marker CD69 in CD8 T cell populations and the Th1 polarization marker CXCR3 in CD4 T cell populations. MPL treatment significantly increased production of Th1-associated, IgG2c anti-tumor antibodies which were required for and predictive of anti-tumor response to RT and MPL and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic anti-tumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4⁺ but not CD8⁺ T cells. <h3>Conclusion</h3> We report the potential for MPL to augment the in situ vaccination effect of combination RT+C4 through FcγR, macrophage, and CD4 dependent mechanisms. To our knowledge this is the first report describing generation of a CD8⁺ T cell-independent, Th1 polarized, systemic anti-tumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

Local TLR4 stimulation augments in situ vaccination induced via local radiation and anti-CTLA-4 checkpoint blockade through induction of CD8 T-cell independent Th1 polarization

BackgroundRadiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced...

Abstract 2908: PT217, an anti-DLL3/anti-CD47 bispecific antibody, exhibits anti-tumor activity through novel mechanisms of action

Abstract Small cell lung cancer (SCLC) is the most lethal subtype of lung cancers with few patients surviving more than 5 years from diagnosis. While SCLC initially responds well to standard chemotherapy treatment, chemotherapy-resistant recurrence is characteristic and available treatment options for recurrent and refractory diseases are limited. Immunotherapy as an alternative or additional treatment has been very successful in treating many cancers, yet it has shown disappointingly limited benefits in SCLC and notable treatment related toxicities have been observed. Delta like 3 (DLL3) is an attractive target for SCLC immunotherapy, as its expression is highly restricted to SCLC and can therefore be used for targeting purposes. On the other hand, CD47 is also strongly expressed on the surface of SCLC cells, where it inhibits macrophage-mediated tumor cell killing. We have developed PT217, a DLL3/CD47 bispecific antibody with effector effect competent Fc to specifically target SCLC cells via two mechanisms. First, PT217 blocks the CD47-SIRPα interaction and stimulates phagocytosis of tumor cells by macrophage. Second, PT217 exhibits robust effector effect (ADCC by NK cells and ADPC by macrophage) mediated by the functional Fc of PT217. Interestingly, it appears that the tumor cells targeted by NK cells are subsequently engulfed by macrophage. Elevated phagocytosis of the tumor cells by macrophages and dendritic cells is expected to increase the presentation of tumor neoantigens that may eventually lead to T cell-mediated killing of tumor cells that do not expression DLL3. In vivo, PT217 demonstrated tumor suppression activity in xenograft mouse models. In addition to the strong tumor killing activity, PT217 also demonstrated good safety profile in NHP studies. Furthermore, PT217 exhibits regular mAb-like manufacturability in process development and CMC. We expect to initiate a phase 1 trial to target SCLC in the first half of 2022. Citation Format: Haiqun(John) Jia, Dorthy Fang, Molly Lobsinger, Yan Long, Kelan Chen, Albert Lam, Yangyang Guo, Kaisa Selesniemi, Ming Wang, Hui Zou. PT217, an anti-DLL3/anti-CD47 bispecific antibody, exhibits anti-tumor activity through novel mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2908.

Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions

ABSTRACT Osteosarcoma metastasizes to the lung, and there is a link between the predominance of tumor-promoting immunosuppressive M2 macrophages in the metastases and poor patient survival. By contrast, M1 macrophage predominance correlates with longer survival. M2 macrophages can be induced by various stimuli in the tumor microenvironment, including exosomes, which are 40- to 150-nm vesicles that are involved in intercellular communication and contribute to tumor progression and immune evasion. Recognizing that tumor cells can influence the tumor microenvironment to make it more permissive and because of the link between M2 dominance and curtailed patient survival, we evaluated the effect of exosomes from non-metastatic K7 and Dunn osteosarcoma cells and the metastatic sublines K7M3 and DLM8 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophages with K7M3 and DLM8 exosomes induced expression of IL10, TGFB2, and CCL22 mRNA (markers of M2 macrophages) and decreased phagocytosis, efferocytosis, and macrophage-mediated tumor cell killing. In contrast, exosomes from non-metastatic K7 or Dunn cells did not inhibit phagocytosis, efferocytosis, and macrophage-mediated cytotoxicity or induce increased expression of IL10, TGFB2 or CCL22 mRNA. In addition, metastatic osteosarcoma cell exosomes significantly increased the secretion of TGFB2, a key signaling pathway associated with tumor- mediated immune suppression. Finally, the inhibition of TGFB2 reversed the suppressive activity of alveolar macrophages exposed to metastatic osteosarcoma cell exosomes. Our data suggest that the exosomes from metastatic osteosarcoma cells can modulate cellular signaling of tumor-associated macrophages, thereby promoting the M2 phenotype and creating an immunosuppressive, tumor-promoting microenvironment through the production of TGFB2.

Exosomal communication by metastatic osteosarcoma cells contributes to the modulation of alveolar macrophages to an M2 tumor-promoting phenotype

Abstract Osteosarcoma (OS) metastasizes to the lung and there is a link between tumor-promoting M2 macrophage content and poorer patient survival. Conversely, high M1 content correlates with improved survival. M2 macrophages are immunosuppressive, contribute to tumor progression and metastasis, and can be induced by various stimuli in the tumor microenvironment, including exosomes (Exo). Exo are small 40–150 nm vesicles that are involved in inter-cellular communication, and contribute to tumor progression, increased tumor survival and immune evasion. Because of the link between M2 content and inferior patient survival, we evaluated the effect of Exo from non-metastatic K7 OS cells and the metastatic subline K7M3 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophage cells with K7M3 Exo induced mRNA expression of IL10, TGFB2, IL1rα, and CCL22, (markers of M2 macrophages), and decreased phagocytosis, efferocytosis and macrophage-mediated tumor cell killing. In contrast, Exo from non-metastatic K7 or 3T3 normal fibroblast cells did not inhibit phagocytosis, efferocytosis or macrophage-mediated tumor cell killing. Our data suggest that the Exo from metastatic OS cells can modulate cellular signaling of tumor associated macrophages, thereby promoting the M2 phenotype and inhibiting their phagocytic and cytotoxic abilities. Identifying the factors within Exo from metastatic OS cells and the mechanisms responsible for Exo communication between metastatic OS cells and macrophages could reveal new therapeutic targets and opportunities to improve the treatment of patients with metastatic disease in the lung.

Correlation between apoptosis and tumor-associated macrophages in human invasive ductal breast carcinoma

The relationship between apoptosis and tumor-associated macrophages (TAM) was studied in situ in 60 breast carcinomas (18 G1, 28 G2 and 14 G3 carcinomas) using simultaneous apoptosis (TUNEL method) and macrophage staining (anti-CD68 antibody). Apoptotic tumor cell rate (ATCR), total TAM content (TAM(TOT)) and the proportion of TAM that had either cell-to-cell contact with apoptotic tumor cells or that were phagocytosing them (TAM/APO cells) were quantified. ATCR correlated significantly with TAM(TOT). Within all apoptotic tumor cells, the proportion of TAM/APO cells was lower than 20%. Considering the fact that cell-to-cell contact is essential for macrophage-mediated tumor cell killing, our data suggest that the majority of apoptoses occurring in breast cancer may not be caused by macrophage tumoricidal activity. TAM/APO cells accounted for only 1.7% of all TAM. Thus, tumor cell killing and apoptotic tumor cell phagocytosis seem to be quantitatively less important functions of TAM in human breast cancer in vivo.

Direct Activation of Murine Peritoneal Macrophages for Nitric Oxide Production and Tumor Cell Killing by Interferon-γ

Interferon-gamma (IFN-gamma) is known to prime macrophages for tumor cell lysis and nitric oxide (NO) production as measured by enhanced sensitivity to lipopolysaccharide (LPS). In the present study, the ability of IFN-gamma to directly activate peritoneal macrophages from C57BL/6 and Balb/c mice for tumor cytotoxicity and NO production was evaluated. Macrophage-mediated tumor cell killing was measured by an 18 h 51Cr release assay using P815 mastocytoma cells as targets. Concurrent NO production was measured as nitrite in the supernatants of macrophage cultures. Incubation of macrophages with IFN-gamma resulted in activation of macrophages for tumor cell lysis. IFN-gamma alone also activated macrophages for NO production under identical conditions. Addition of LPS along with IFN-gamma resulted in synergism in the activation of macrophages for both cytolysis and NO production. LPS contamination of the IFN-gamma preparation was absent as evidenced by the following criteria: (1) the IFN-gamma preparation as well as the reagents used were shown to be free of LPS contamination based on LAL endotoxin tests (sensitivity 25 pg/ml), (2) the ability of IFN-gamma to activated macrophages was not abrogated by prior treatment of the cytokine with polymyxin B, whereas the effect of LPS was inhibited (70-100%) under similar conditions, (3) pretreatment of the IFN-gamma preparation with a specific endotoxin neutralizing protein did not abrogate the ability of IFN-gamma to induce macrophage activation, and (4) heat treatment of solutions containing IFN-gamma alone or IFN-gamma+LPS abolished only the effect of IFN-gamma, not that of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cell granules inhibit macrophage-mediated lysis of mastocytoma cells (P815) and nitric oxide production.

The effects of mast cell granules (MCGs) on macrophage-mediated lysis of P815 mastocytoma cells and nitric oxide (NO) production were studied. Murine peritoneal macrophages exhibited tumor cell killing and NO production only when activated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma). Coincubation of macrophages with MCGs during LPS activation dose-dependently inhibited macrophage-mediated tumor cell lysis. The MCG effect was not due to inactivation or removal of LPS by MCG. The inhibitory effect was also not due to histamine or serotonin present in the MCGs. The granules were not toxic to macrophages or P815 mastocytoma cells. The effect of MCGs on macrophage-mediated tumor cell killing was evident whether MCGs were added before or after a 4-h exposure of macrophages to LPS. However, the inhibitory effect was not seen if MCGs were added after macrophages had been exposed to LPS for 24 h. To assess whether MCGs could inhibit a non-LPS trigger, MCGs were tested on macrophages activated with IFN-gamma. In these experiments, MCGs dose-dependently inhibited macrophage-mediated tumor cell killing induced by IFN-gamma, LPS, or IFN-gamma plus LPS. Furthermore, in parallel experiments, MCGs significantly inhibited macrophage NO production induced by LPS, IFN-gamma, or IFN-gamma plus LPS. Pretreatment of MCGs with diisopropylfluorophosphate, a serine protease inhibitor, only partially abrogated the effects of MCGs. The results demonstrate that MCGs inhibit both LPS- and IFN-gamma-induced macrophage killing of P815 cells and the inhibition is associated with the decrease of NO production.

Mechanisms of macrophage‐mediated tumor cell killing: A comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor

The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.

Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins).

Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes.

Spontaneous Tumor Cell Killing by Human Blood Monocytes and Human Peritoneal Macrophages: Lack of Alteration by Endotoxin or Quenchers of Reactive Oxygen Species&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Human mononuclear phagocytes (monocytes and macrophages) act as effectors in the destruction of tumor cells. Peritoneal macrophages from normal or infertile women killed a variety of tumor cells in vitro more efficiently than did blood monocytes from the same subjects. Lysis depended on the effector-to-target cell ratio and was neither reproduced by supernatants from nor lysates of the mononuclear phagocytes. Normal fibroblasts were not lysed. Lipopolysaccharide (10(1)-10(4) ng/ml) did not alter the monocyte- or macrophage-mediated tumor cell killing. The monocytes and macrophages had equivalent basal and phorbol 12,13-myristate acetate-stimulated H2O2 and O-2 production, and the reactive oxygen species scavengers or quenchers catalase, superoxide dismutase, mannitol, and L-histidine did not diminish the killing. These observations suggest that the spontaneous tumor cell killing by human mononuclear phagocytes was not mediated by reactive oxygen species.

A photometric and plaque assay for macrophage mediated tumor cell cytotoxicity

Pretreatment of L-929 cells for 2–3 h with 2 μg/ml actinomycin D followed by washing rendered them exceedingly sensitive to the direct cytotoxic effects of murine and rabbit macrophages. We exploited the tremendous increase in sensitivity of L-929 cells to effector cell mediated cytotoxicity by demonstrating a simple and convenient photometric and plaque assays capable of being completed in 18 h for the quantitation of macrophage mediated tumor cell killing. The plaques demonstrated were generally visible to the unaided eye and were linearly related to the number of effector cells plated indicating that a plaque represented multitarget cytolysis attributed to a single effector cell. Unlike previously described assays, the effector: target ratios demonstrated were extremely low. Using the described techniques, it was estimated that a single macrophage could kill from approximately 10 to 1000 actinomycin D pretreated and washed target cells. In the presence of LPS-activated effector cells, the majority of these targets that stained with eosin Y did so in a cluster pattern, indicating cytotoxicity rather than mere detachment from the monolayer surface.

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE 2 and PGI 2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E 2 (PGE 2) and prostaglandin I 2 (PGI 2), the latter detected as its stable metabolite, 6-keto PGF 1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10 −7M for PGI 2 and 3 × 10 −8M for PGE 2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE 2 and PGI 2; however, only PGE 2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.

Macrophage-mediated tumor cell killing: regulation of expression of cytolytic activity by prostaglandin E.

The expression of nonspecific macrophage-mediated tumoricidal activity in vitro is a transient phenomenon. The work reported here shows that the loss of tumoricidal activity by mouse macrophages is attributable, at least in part, to the effects of prostaglandin E (PGE). The amount of PGE needed to inhibit the expression of cytolytic activity (2 x 10(-9) M) was readily synthesized and secreted by the same population of resident peritoneal macrophages that had been induced to kill tumor cells by exposure to 100 ng/ml bacterial lipopolysaccharide (LPS). Inhibitory concentrations of PGE were present in supernatants within the 1st hr after macrophages were exposed to LPS. In spite of this fact, cytolytic activity still developed, and 12 to 16 hr were required for the PGE to have its full inhibitory effect. Thus, PGE did not act by blocking the development of cytolytic activity. Treatment of LPS-stimulated macrophages with 10(-6) M indomethacin (or other cyclooxygenase inhibitors) prevented both PGE synthesis and the shut-off of cytolytic activity. The latter effect could be reversed by adding PGE2 to the cultures at a concentration of 10(-8) M. Lastly, PGE inhibited cytolytic activity mediated by indomethacin-treated macrophages only when activator was present: pulsing with PGE before the addition of LPS had a negligible inhibitory effect. These findings may help explain why intratumoral macrophages often lack nonspecific cytolytic capabilities.

Bacterial lipopolysaccharides and mycoplasmal lipoglycans: A comparison between their abilities to induce macrophage-mediated tumor cell killing and [formula omitted] amebocyte lysate clotting

The ability of various bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) to induce macrophage-mediated tumor cell killing and Limulus amebocyte lysate clotting was determined. Lipoglycans from the mycoplasma Acholeplasma axantum or Acholeplasma granularum had no activity or 10 4 to 10 5 less activity than lipopolysaccharides from Escherichia coli 0128:B12, Escherichia coli K235, or Salmonella minnesota R595 in causing Limulus lysate clotting and tumor cell killing by peritoneal macrophages from normal or bacillus Calmette-Guérin-infected mice. Previous studies have shown that the lipid A portion of bacterial lipopolysaccharide is responsible for the effects on macrophage-mediated tumor cell killing and Limulus lysate clotting. The known differences in the lipid structures of bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) may account for the noted differences in the biologic potencies observed here.

Macrophage-Mediated Tumor Cell Killing: Lack of Dependence on the Cyclooxygenase Pathway of Prostaglandin Synthesis

M phi obtained directly from disaggregated murine Moloney sarcomas produced PGE2 and a hydroxy fatty acid derivative as the major products of arachidonic acid metabolism. M phi-immunoreactive PGE synthetic rates decreased substantially and cytotoxic activity was lost when freshly explanted tumor M phi were held in culture 24 hr. Such cultured M phi remained in a partially activated "primed" state, however, wherein the addition of minute (ng) amounts of bacterial lipopolysaccharide (LPS) returned cytolytic activity and PGE synthesis to original levels. Indomethacin-induced blockade of the M phi cyclooxygenase pathway inhibited PG synthesis by LPS-stimulated, primed M phi without affecting the return of cytolytic activity. We conclude, therefore, that the production of PG had no direct role in the mediation of tumor cell killing by activated M phi isolated from these neoplasms.

Characterization of the Effects of Endotoxin on Macrophage Tumor Cell Killing

Abstract These studies demonstrate the potent effect of LPS on the induction of macrophage-mediated tumor cell killing and show, by using pure populations of cloned macrophages as effector cells, that LPS can act in the complete absence of contaminating lymphocytes. Macrophage differentiation toward the tumoricidal state parallels the responsiveness of macrophages to LPS. Normal macrophages will not kill even in the presence of 10 µg/ml LPS; peptone-induced normal macrophages (stimulated macrophages) are made tumoricidal by ≥500 mg/ml LPS; and nontumoricidal BCG-activated macrophages are made to kill tumor cells by 0.5 to 1.0 ng/ml LPS. The LPS effect is reproduced by lipid A. The effect is abolished by alkaline treatment of LPS or by the presence of polymyxin B. Nontumoricidal BCG-activated macrophages from the lipid A-nonresponder mouse strain C3H/HeJ require 250 times more LPS to become tumoricidal than do nontumoricidal BCG-activated macrophages from the C3H/HeN strain. LPS acts synergistically with MAF-rich peritoneal cell supernatants in causing macrophages to kill tumor cells. Although LPS can elicit MAF production by lymphocytes, it can also act directly on cells of lymphocyte-free macrophage colonies to render them tumoricidal. Reagents are frequently contaminated with LPS, and unless this is noted, valid interpretation of experiments investigating macrophage tumor cell killing is difficult. Means of detecting, detoxifying, and eliminating the LPS of experimental agents are discussed.