Methods for ablating neurons.

This past year, laser ablation has been applied to investigations of neuromuscular connectivity in Drosophila, neuronal function in nematode, and mammalian central nervous system development. Ablation by targeted gene expression has been refined and applied to questions of neural development. Chromophore-assisted laser inactivation has been used to demonstrate distinct functions for two proteins during grasshopper neural development.

Characterization of effector functions of v-raf/mil- and v-myc-transformed murine splenic macrophage cell lines.

We have characterized several of the cytocidal effector functions of a series of cell lines derived by recombinant retroviral transformation of individual clones of C3H/HeJ mouse splenic macrophages. The three cell lines described in this report (4.01, 4.07, 4.14) all expressed equivalent tumoricidal activity against P815 tumor target cells. However they differed in their high avidity binding of tumor cells (4.01 = 4.14 greater than 4.07), as well as in the killing of Leishmania major (4.01 = 4.07 greater than 4.14), the expression of antibody-dependent cellular cytotoxicity against chicken erythrocytes (4.14 greater than 4.01 greater than 4.07), and finally, in the tumor-stimulated release of tumor necrosis factor-alpha (4.01 = 4.14 greater than 4.07). The stable and restricted expression of distinct effector functions among these three cell lines makes them particularly valuable as models for establishing the precise mechanisms by which cytocidal functions are effected. In addition, they should also prove of value in understanding the basis for macrophage functional diversity.

Characterization of tumor binding by the IC-21 macrophage cell line.

The purpose of this study was to determine if the SV40-transformed murine macrophage cell line IC-21 is a suitable model to study the selective high avidity binding of tumor cells by subpopulations of activated macrophages. IC-21 macrophages bound P815, RBL5, and EL-4 murine tumor cells with high avidity, as measured by the inverted centrifugation method. Tumor binding by IC-21 macrophages was competitively inhibited by crude membrane vesicles prepared from tumor cells but not by cell membranes prepared from nontransformed splenic leukocytes, suggesting that this process was mediated by tumor-specific binding sites. IC-21 macrophages and primary cultures of pyran copolymer-elicited peritoneal macrophages demonstrated similar tumor binding avidity, kinetics, saturability, and metabolic requirements for optimal high avidity tumor binding. However, compared with primary cultures of pyran copolymer-elicited peritoneal macrophages, IC-21 macrophages bound 4-fold more tumor cells and were more homogeneous for tumor binding capability. Finally, one third of maximal tumor cell binding by IC-21 macrophages was completed within 5 min of contact with tumor, suggesting that IC-21 macrophages constitutively expressed some high avidity tumor binding sites. Their stable and homogeneous capability for binding tumor cells and their ease of growth make the IC-21 macrophage cell line a potentially valuable model for elucidating the molecular mechanisms responsible for selective high avidity tumor binding by subpopulations of activated macrophages.

Macrophage tumor necrosis factor-alpha release is induced by contact with some tumors.

The purpose of this study was to determine whether macrophages were directly stimulated by tumor cells to release TNF-alpha. We found that several murine and human tumor cell lines and crude cell membrane vesicles prepared from these tumor cells stimulated pyran copolymer-elicited murine peritoneal macrophages (PEM) to release as much as 362 +/- 69 (mean +/- SE) units of TNF activity per 10(6) PEM in vitro. By contrast, several nontransformed cells, including Con A-stimulated splenic leukocytes and CTLL cloned T lymphocytes, failed to stimulate PEM to release TNF. Antibody and complement-mediated depletion of macrophages abrogated the release of TNF; whereas depletion of NK cells and T lymphocytes did not affect tumor-stimulated TNF release, suggesting that tumor cells directly stimulated PEM to release TNF. Tumor-stimulated TNF release was rapid, peaking in 2 to 3 h with subsequent loss of TNF activity from the medium. In the absence of tumor, PEM contained detectable levels of TNF mRNA, but did not release functionally active TNF. The addition of P815 tumor cell membrane vesicles increased both TNF mRNA levels, peaking at 1 to 2 h, and release of high levels of TNF activity. Confounding effects of endotoxin were excluded by the resistance of tumor-stimulated TNF release to neutralization by polymixin B, and by the equivalent responsiveness of PEM from endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive (C3H/HeN) mice to stimulation by tumor cells. Factors which stimulated PEM to release TNF could be extracted from tumor cell membrane, with 77% of the macrophage-stimulating activity recoverable in aqueous phase. In conclusion, we have demonstrated that some tumor cell lines express specific characteristics which can be recognized by macrophages and which stimulate macrophages to release TNF.