It is well known that activated macrophages generate a variety of cytotoxic factors, oxygen radicals, and nitric oxide. Nitric oxide is considered the major effector against tumoral cells. Even though activated macrophages are efficient tumoral cell killers, this macrophage property is abridged in some patients and experimental animals bearing malignant tumors. We previously observed that in L5178Y tumor-bearing mice, the macrophages converging at the ascites tumor did not exhibit tumoricidal activity against malignant lymphoblasts. However, after the tumoral cells were killed or severely injured (as a result of a chemotherapeutic experimental schema), the associated macrophages from the ascites tumor became activated against L5178Y lymphoblasts. In addition, Wiltrout et al. showed that tumor-associated macrophages recovered from L5178Y lymphoma-bearing mice conserve their entire capacity and could be activated with LPS-killed malignant lymphoblasts in vitro. The previously mentioned actions strongly suggest that intact tumoral cells release certain molecules that reversibly inhibit macrophages from tumor-bearing animals. Accordingly, the objectives of the present study were the following: (a) to determine whether the activation of macrophages with LPS, IFN-?, or a combination of both were prevented by mouse cell-free ascitic liquid (CFAL) or the soluble fraction (S1) from L5178Y lymphoblast lysates, and (b) to investigate whether macrophages from healthy or tumor-bearing mice were susceptible to inhibition by CFAL or S1.

The L5178Y murine cell line was used in this study. This is a transplantable murine leukemia cell line, which was derived in 1952 by LW Law from a thymic tumor induced in a DBA/2 mouse by methylcholanthrene. This cell line has been maintained for the last 20 years in our laboratory by serially inoculating the lymphoblasts into BALC/c mice (see subsequent text).

Two-month-old male BALB/c mice were used to both maintain the tumoral cell line and as a source of normal- and tumor-bearing peritoneal macrophages. The animals were kept in polypropylene cages with food (Lab Diet 5001, PMI Nutrition International, Inc., Brentwood, MO, USA) and water ad libitum under controlled conditions of light, humidity, and temperature.

The mice were serially inoculated as follows: one healthy male BALB/c mouse, 2 months of age, was inoculated intraperitoneally with 100 ?L of ascites liquid containing 2 × 10⁷ lymphoblasts. Twelve days later, the ascitic liquid was harvested and used to inoculate a new mouse, and so on.

Four mice were injected i.p. with 2 mL of 4% thioglycollate (Merck de México, Mexico City, Mexico) in phosphate-buffered saline; (K₂HPO₄ 0.02 M, KH₂PO₄ 0.02 M, NaCl 0.12 M, pH 7.4; PBS). Ninety-six hours later, the macrophages were harvested by peritoneal cavity lavage with RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA). The liquid obtained was centrifuged at 450 × g for 10 min at room temperature. The supernatant was discarded and the cellular pellet resuspended in 4 mL of RPMI-1640 fresh medium. Each of the previously mentioned materials was placed on a 4-mL NycoPrep^™ 1.068 layer (Nycomed Pharma Centrifugation Media, Oslo, Norway) and the preparation was centrifuged at 600 × g for 10 min at room temperature. The macrophages were recovered from the ascites lymphoma liquid with NycoPrep^™ 1.068 as recommended by the manufacturer. In brief, 4-mL ascites lymphoma liquid was placed in a 13 × 100 mm glass assay tube containing a 4-mL NycoPrep^™ 1.068 layer. This preparation was centrifuged at 600 × g for 10 min at room temperature. Thereafter, three bands were formed in the tube. The central tube, placed just above the pellet, contained the macrophages. To recover the macrophages, the upper layer (3–4 mm wide) was extracted with a vacuum and discarded. The layer below was carefully extracted with a Pasteur pipette, placed into a clean assay tube, and two volumes of fresh RPMI-1640 medium were added and centrifuged as before. The supernatant was discarded and the macrophage pellet was resuspended and washed twice with RPMI-1640 medium. A sample from these cells was dyed with Wright’s stain, and the cells’ major monocyte/macrophage characteristics were verified as follows: reniform or fusiform cells sized 15–20 ?m with a dark violet-colored nucleus and one or two nucleoli, showing light-blue vacuolated cytoplasm with multiple, fine pink-purple granules. This preparation density was brought to 1 × 10⁶ cells/mL with RPMI-1640 medium plus 10% fetal bovine sera. Afterward, each of the 96 wells from a microplate (Sigma Chemical Co.) was inoculated with 200 ?L of the previously mentioned cell suspension and the microplate was incubated at 37°C in a 5%-CO₂ atmosphere for 24 h.

Two milliliters of ascitic liquid was harvested by aspiration from a 12-day-old tumor-bearing mouse. The liquid was placed in a 13 × 100 mm glass assay tube containing 4 mL of NycoPrep^™ 1.068 layer. The macrophage band was saved and the cells washed twice with RPMI-1640 medium. Monocyte/macrophage lineage was confirmed by Wright staining, as carried out with normal macrophages. Cell number was determined with a hemocytometer, and their density adjusted to 1 × 10⁶ cells/mL with RPMI-1640 medium plus 10% fetal bovine serum. Subsequently, 200 ?L of the lymphoma cell suspension were poured into each of the 96 wells of a microplate and incubated at 37°C in 5%-CO₂ atmosphere for 24 h.

The ascitic liquid from two mice bearing a 12-day lymphoma was pooled (4 mL) and centrifuged at 600 × g for 10 min at room temperature. The supernatant (CFAL) was saved and stored at ?20°C until used. The cell pellet was mixed with a 4-mL lysis solution (1 mM EDTA, 1 mM epsilon amino caproic acid, and 1 mM dithiothreitol [Calbiochem, San Diego, CA, USA]). This preparation was frozen at ?20°C for 24 h and thawed at room temperature. The suspension was shaken vigorously with a vortex and centrifuged at 600 × g/10 min. The supernatant (S1) was stored at ?20°C until used.

The microplates containing adherent macrophages were incubated in the presence of 5 ?g/mL lipopolysaccharides (LPS), 40 U/mL IFN-? (Sigma Chemical Co.), or a LPS (5 ?g/mL)/ IFN-(? 40 U/mL) blend. The quantity of nitric oxide released by the macrophage cultures was quantified in their cell-free supernatant after 48 h of incubation in the presence or absence of tumor fractions according to the Griess method12. D.J. Stuehr and C.F. Nathan, Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169 (1989), p. 1543. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (867). Optical density measurements at 545 nm were carried out with a microplate reader (ElA Multi-Well Reader, Sigma Diagnostic (St. Louis, MO, USA). All determinations were done in triplicate.

MDA was assayed as follows: the macrophage cultures (as described in Materials and Methods), were supplemented with 5 ?g LPS/mL, 40 U IFN-?/mL, or a combination of 5 ?g LPS U/mL and 40 IFN/mL; these preparations were incubated at 37°C for 24 h; 10 ?L of CFAL or S1 were added; the plates were reincubated for 24 h, and the amount of nitric oxide determined in the spent culture medium.

MAIA assays were carried out as follows: the macrophage cultures were added with 10 ?L of CFAL or tumor fractions and incubated at 37°C; after 24 h at 5 ?g LPS/mL, 40 U IFN-?/mL, or a combination of 5 ?g LPS U/mL and 40 IFN/mL were added and reincubated at 37°C for 48 additional hours, and the amount of nitric oxide released by macrophages was determined in their spent culture medium.