Two‐color flow cytometric analysis of the expression of MAC and MHC Class II antigens on macrophages during tumor growth

Abstract

Tumor-bearing host (TBH) macrophages (M+) exhibit immune dysfunction that is concomitant with phenotypic changes. We examined M+ subpopulations by changes in the expression of surface antigens Mac-1, -2, -3, and Ia on normal and TBH peritoneal and splenic M+. M+ were double-labeled and analyzed by flow cytometry to observe multiple expression of surface antigens. Tumor growth alters the multiple expression of these M+ markers. Peritoneal and splenic M+ had different Mac+ and Mac+Ia+ population percentages. In TBH, peritoneal M+ had decreased percentages of Mac-1+2+, Mac-1+3+, Mac-2+3+, and Mac+Ia+ M+. This decrease correlated with functional changes in TBH M+. In contrast, there was an increase in Mac-2-Ia-TBH peritoneal M+. Previously undiscovered Mac-1+2-3-and Mac-1-2-3+ populations were found. In contrast to peritoneal M+, there was an increase in the percentage of Mac-l+2+, Mac-1+3+, and Mac-2+3+ splenic TBH M+ but, like peritoneal M+, there was a decrease in the percentage of Mac+Ia+ M+. Also, TBH splenic M+ showed a smaller but more uniform antigen density than normal host splenic M+. Tumor growth modulated phenotypic alterations in peritoneal and splenic M+ subpopulations. Combined with earlier functional studies of M 4 subpopulations, these data suggested a relationship between changes in M+ phenotype and tumor-induced dysfunction of M+-modulated immune activity.Key terms: Macrophage surface antigens, double-label techniques, monoclonal antibodies, Mac-1, Mac-2, Mac-3, Ia, tumor-bearing host Macrophages (M+) have diverse functions that may reflect the existence of distinct subpopulations of M+ (9-11,16,29-31,341. To understand M+ functions during the immune response, these subpopulations need to be characterized and their functional relationships established. Subpopulation delineation is critical in studies involving tumor-bearing hosts (TBH), because tumor growth changes the immunoregulatory properties of M+ (6)(7)(8)(9)(10)(11)(15)(16)(17)(18)24,25,28,33,35,36) and changes also occur in M+ phenotype and function. Phenotypic changes in TBH M+ include changes in surface antigen (Mac-1, Mac-2, Mac-3, and Ia) expression (9)(10)(11)16,19,25,34), size distribution (341, and peroxidase staining (11). Concomitant functional changes in TBH M+ include decreased accessory cell function in the allogeneic mixed lymphocyte reaction (MLR) (6,10,11,16,24), the autologous mixed lymphocyte reaction (AMLR) (8,35,36), and mitogen-induced T cell proliferation (7,9,10,25,33). These functional changes result from an altered suppressor M+ population (9,11,16,25,(34)(35)(36) and a decreased production of enhancing factors (18,28,35). If the phenotype of the suppressor M+ could be identified (reported here) and then correlated with its specific function, a greater understanding of the mechanism of TBH M+-mediated control of immune reactivity would emerge.

Tumor-induced immune dysfunction: the macrophage connection

“…Spleens normally have high numbers of class II ϩ Ms, which produce far less TNF-␣ and PGE 2 [118,119], and perhaps NO, than do class II Ϫ Ms. As expected, splenic Ms are much better antigen-presenting cells than are peritoneal Ms because splenic Ms express more MHC class II molecules [3,137,154,196]. Antigen presentation is drastically compromised in the TBH spleen because of increased numbers and suppressor activity of class II Ϫ Ms [3,137,156].…”

“…In addition, tumors can affect changes in M phenotype and thus favor a particular function. Using depletion [12,136,192,193] and single [154,[193][194][195] and double-label [195,196] fluorescent antibody studies, we showed a shift in M subpopulations, changes in M marker expression, and changes in M function during tumor growth.…”

“…There are sufficient numbers of antigen-presenting class II ϩ splenic Ms in the TBH because suppression of T cell proliferation is reversed when suppressor molecule activity is blocked [201]. The investigations of M class II expression and factor production during tumor growth, using a nonmetastatic tumor located away from the peritoneum or spleen [154,196], showed increased production of TNF-␣ and NO by splenic Ms that still remains less than that produced by peritoneal TBH Ms [120]. Tumors that metastasize to the spleen, therefore, may have a better chance of survival than do those in inflammatory sites where exudate Ms are active.…”

“…Tumor growth increases the numbers of splenic class II Ϫ Ms [196] and their suppressor and cytotoxic activities [118,122,202,205]. Although increased splenic class II Ϫ M suppressor and cytotoxic activities are mediated by PGE 2 and TNF-␣ [118,119,122], respectively, NO and H 2 O 2 also may contribute to these activities, as do peritoneal class II Ϫ Ms [5,7,206] and monocytes [125].…”

“…Tumor growth increased the number of class II Ϫ Ms in the spleen from roughly 30% to 70% [154,196]. If class II Ϫ Ms are a prime cause of immunosuppression, regulation of class II expression may be a major mechanism through which tumors exert influence over the immune system.…”