Ronald E. Garner

Secretion of TNF-alpha by alveolar macrophages in response to Candida albicans mannan.

Resident alveolar macrophages (AMø) were tested for their ability to respond to Candida albicans mannan. AMø were found to produce tumor necrosis factor alpha (TNF‐α) in vitro in response to mannan stimulation. TNF‐α secretion was measured using ELISA and L929B cellular cytotoxicity assays. Cytotoxicity was neutralized in parallel L929B cell cultures by the addition of rabbit anti‐TNF‐α antibody. Mannan preparations were found to be free of contaminating LPS by Limulus assay. When AMø were cultivated for 18 h at 37°, 67 μg of mannan stimulated the secretion of approximately 207 U/ml of TNF‐α. By comparison, AMø treated with 6.7 μg of LPS secreted approximately 257 U/ml of TNF‐α. Optimal TNF‐α production occurred between 9 and 18 h after mannan stimulation. Disparate mechanisms for stimulation of TNF‐α secretion were suggested by differential sugar blockade of LPS‐ and mannan‐induced TNF‐α secretion. The addition of 2% D‐mannose or 2% alpha‐methyl‐D‐mannoside to AMø cultures blocked mannan‐but not LPS‐stimulated TNF‐α secretion. Furthermore, the addition of rabbit anti‐mannan antibody to mannan‐coated plastic culture dishes prevented TNF‐α secretion by the mannan‐sensitive RAW 264.7 cell line. Moreover, the data suggest that C. albicans mannan stimulated AMø to secrete TNF‐α by an LPS‐independent receptor mechanism which may also function as a mannose receptor. J. Leukoc. Biol. 55: 161–168; 1994.

Shifts in macrophage (Mφ) surface phenotypes during tumor growth: association of Mac-2+ and Mac-3+ Mφ with immunosuppressive activity

Rat anti-mouse monoclonal antibodies (mAb), anti-Mac-1, -2, and -3, directed against macrophage (M phi) glycoprotein surface antigens, were used to demonstrate a tumor-induced shift in peritoneal M phi subpopulations. This study of the tumor-induced shift was approached in two steps. First, to show that separate phenotypic M phi subpopulations existed and second, to show that a shift in these populations was involved in immunosuppression of the host during tumor growth. Endogenous peroxidase activity was examined among normal and tumor-bearing host (TBH) M phi. A significant increase in the number of peroxidase-positive M phi occurred during tumor growth. Indirect immunofluorescence showed a decrease in Mac-2+ cells and an increase in Mac-3+ cells in TBH M phi populations. When the mAb, anti-Mac-1,-2, and -3 were used in the presence of complement (C), they were cytotoxic for M phi and showed differential depletion of normal and TBH M phi. Peroxidase-positive TBH M phi were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not by anti-Mac-2, whereas no direct relationship was observed among normal host M phi. To demonstrate differences between normal and TBH M phi subpopulations, soluble inhibitory factors were examined from mAb plus C-modified M phi populations. Anti-Mac plus C-treated normal and TBH M phi produced supernatants with different regulatory capabilities as assessed in the mixed-lymphocyte reaction (MLR). Anti-Mac-2 plus C treatment significantly reduced the ability of TBH M phi to produce a soluble suppressor(s) but did not alter normal host M phi-derived suppressor production. In contrast, anti-Mac-1 and -3 plus C treatment of normal host M phi significantly reduced suppressor production. In the TBH, however, anti-Mac-1 plus C had no effect, while anti-Mac-3 plus C had only a limited reduction as compared to the normal host. Determination of levels of prostaglandin E2 (PGE2) in M phi supernatants showed that normal host Mac-1+ M phi were involved in down regulation of PGE2 production. This control was missing in the TBH M phi. Mac-2+ M phi were the apparent producers of PGE2 which accounts for the factor-mediated MLR suppression attributed to TBH Mac-2+ M phi. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can in part be attributed to differences in anti-Mac mAb-defined M phi subpopulations.

Prostaglandin E2 production by Mac-2+ macrophages: tumor-induced population shift.

Tumor growth induced a shift in the phenotype of macrophages (MΦ) responsible for factor‐mediated suppression of allogeneic mixed lymphocyte reactions (MLR), and the suppression by tumor‐bearing host (TBH) Mac‐2+ MΦ was in part due to production of prostaglandin E2 (PGE2). Thioglycollate‐elicited peritoneal MΦ from normal and TBH BALB/c mice were modulated with anti‐Mac‐1, ‐2, or ‐3 monoclonal antibodies (mAb) or depleted with mAb plus complement and cultured in the presence or absence of indomethacin. Culture supernatants derived from mAb plus complement‐depleted Mo were added to the MLR at time of initiation and showed that the suppressor phenotype shifted from Mac‐3+ in the normal host to Mac‐2+ in the TBH. Mac‐1+ MΦ also appeared to be involved in suppression by normal host, but not TBH, MΦ. Loss of MLR suppression (increase in MLR reactivity) correlated with an increase in protein content of the culture supernatants. In an effort to explain both this relationship and the mechanism of MLR suppression, PGE2 levels of culture supernatants were determined by radioimmunoassay. Mac‐1+ MΦ were involved in the regulation of PGE2 production in normal hosts, as both activation and depletion caused an increase in PGE2 production. Depletion caused a more dramatic increase in PGE2 production than did activation, suggesting that Mac‐1+ MΦ had a dampening effect on PGE2 production. In contrast, no Mac‐1+ MΦ‐mediated regulatory function occurred in the TBH. Mac‐3+ MΦ were involved in the regulation of PGE2 production in both normal and TBH. Mac‐2+ MΦ were the primary producers of PGE2 in the TBH, but not in the normal host, as their depletion in the TBH caused a significant loss of PGE2 production. Thus, immunosuppression in the TBH was at least partly due to the inability of Mac‐1+ and/or Mac‐3+ MΦ to control production of PGE2 by Mac‐2+ MΦ.

Variations in macrophage antigen phenotype: a correlation between Ia antigen reduction and immune dysfunction during tumor growth.

Variable la antigen expression by macrophages (Mφ) was examined during tumor growth by measuring: i) la antigen masking and immunofluorescence by anti‐la antibody, ii) accessory cell function in concanavalin A (Con A) and mixed lymphocyte reaction (MLR)‐induced T cell proliferation, and iii) Mφ stimulatory function in the MLR. Tumor‐induced progressive loss of la antigen expression was shown by immunofluorescence and corroborated by anti‐la blockade of MLR stimulatory activity of normal but not tumor‐bearing hosts (TBH) splenic Mφ. The TBH splenic Mφ supported Con A‐induced proliferation of syngeneic T cells (la antigen‐independent) but did not support syngeneic T cell proliferation in the MLR (la antigen‐dependent). Irrespective of tissue source, normal and TBH Mφ differed in their MLR stimulatory capabilities. In general, splenic Mφ preparations were better stimulators of allogeneic T cell blastogenesis in the MLR than thioglycollate‐elicited peritoneal Mφ. Kinetic studies with TBH Mφ showed a significant progressive loss in MLR stimulatory activity, which was especially pronounced with peritoneal Mφ. Expression of la antigens by normal but not TBH Mφ were diminished by 24‐h in vitro plating of the peritoneal Mφ. Indomethacin treatment showed Prostaglandin E2 was not a direct in vitro factor in la antigen‐mediated reduction of splenic Mφ MLR stimulatory activity. Taken together, these data delineate a loss of Mφ la antigen expression, resulting in a decrease in la antigen‐mediated functional activities during tumor growth.

Changes in Splenic Macrophage Mac Antigen Expression During Tumor Growth: A Kinetic Study of Accessory Cell Function and Antigen-Defined Phenotypes

Three monoclonal antibodies, anti‐Mac‐1, ‐2, and ‐3, were used to modify accessory cell activity of whole spleen cell (WSC) or splenic adherent cell (SAC) preparations from normal or tumor‐bearing hosts (TBH). Ligand activation by anti‐Mac‐1, ‐2 and ‐3 modified normal and TBH WSC lectin responses differentially; the most pronounced effect was with anti‐Mac 1, which augmented normal WSC responsiveness, whereas anti‐Mac‐2 and ‐3 augmented TBH WSC mitogenesis. Ligand interaction with Mac‐2 epitopes resulted in significantly suppressed normal host WSC responsiveness. With complement, anti‐Mac‐1 and ‐3 each reduced normal and TBH WSC proliferation. Reconstitution of blastogenesis was obtained by combining Mac‐2‐depleted with Macs‐depleted normal host WSC or by combining Mac‐1 ‐depleted with Mac‐2‐depleted TBH WSC. To evaluate the role of different types of splenic adherent cells in T cell lectin responsiveness, adherent cells were collected and depleted by antibody plus complement treatment and added back to normal T cells. Removal of TBH SAC indicated the number of Mac‐1+ SAC susceptible to lysis increased during tumor growth, whereas those susceptible to anti‐Mac‐2 and‐3 treatment decreased. Removal of Mac‐1 + normal host SAC stimulated the supportive accessory function of the remaining SAC. Enhancing accessory cell function diminished after removal of Mac‐2+ or Mac‐1 + normal and TBH SAC, respectively. T cell responsiveness was increased by adding back combinations of normal host SAC, Mac‐1‐depleted with Mac‐3‐depleted SAC or Mac‐1‐depleted with Mac‐2‐depleted SAC but not by Mac‐2‐depleted with Macs‐depleted SAC. In contrast, none of the TBH SAC combinations were completely restored in accessory activity. In summary, SAC from normal host demonstrated an accesssory cell function corresponding to a Mac‐1‐ phenotype, which was either replaced or obscured by the predominance of a Mac‐1+ phenotype in TBH.

Normal and tumor-bearing host macrophage factor-mediated modulation of mixed-lymphocyte reaction responsiveness: separation of T-lymphocyte subset susceptibility to enhancing and inhibitory factors

The mixed-lymphocyte reaction reactivity of normal and tumor-bearing host (TBH) T-cell subsets was examined in response to normal and TBH macrophage (M phi) supernatants. Both inhibiting and enhancing activities were identified in normal and TBH M phi supernatants. The present data suggest that TBH M phi supernatants contained more inhibitory activity than normal host M phi supernatants and that enhancing activity of M phi supernatants was restricted to the Lyt 2,3+ population of cells. TBH Lyt 2,3+ cells were more responsive to the enhancing molecule(s) than their normal counterparts. These data were consistent with studies which implicate M phi as being partially responsible for the immune dysfunction seen in TBH, and extends previous findings on the ability of M phi to regulate the immune response in an attempt to achieve homeostasis.