Andrew D. Yurochko

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.

Changes in Macrophage Populations: Phenotypic Differences between Normal and Tumor-Bearing Host Macro phages

As a tumor grows, changes occur in the function of macrophages (M phi). This is concomitant with changes in their phenotype. Flow cytometric analysis of monoclonal antibody (mAb)-labeled thioglycollate-elicited peritoneal, and resident splenic, M phi showed a tumor-induced shift of Mac-1, -2, -3, and Ia antigen expression. During tumor growth, the percentage of peritoneal Mac-2+, -3+, and Ia+ M phi decreased significantly (22%, 14%, and 58%, respectively), while Mac-1+ M phi remained unchanged. By analyzing the data on two-dimensional histograms and comparing the sizes of M phi to cell-surface antigen expression, we identified distinct subpopulations of peritoneal M phi. Three distinct size versus antigen expression M phi subpopulations were detected by flow cytometry and consisted of 10-16, 17-22, and 23-27 microns for the small-, medium-, and large-sized populations, respectively. Large-sized Mac-1+ and -2+ M phi decreased (37% and 38%), while large-sized Mac-3+ M phi did not decrease during tumor growth. Medium-sized Mac-3+ M phi decreased 33% during tumor growth, while no differences could be seen in medium-sized Mac-1+ or -2+ M phi. Concomitant with the decrease in large-sized Mac-1+ M phi was an increase in small-sized Mac-1+ M phi. Peritoneal Ia+ M phi were mostly small-sized (4-7-fold increase over the medium-sized and none in the large-sized population). M phi Ia antigen expression was nearly absent in the 21-day tumor-bearing host, with less than 4% of the cells labeling positive (a 73% drop from normal host M phi). In splenic M phi, the percentage of Mac-1+ M phi significantly increased (90%) during tumor growth, while Mac-2+ and -3+ M phi showed a smaller, but still significant, increase (48% and 40%, respectively). Additionally, splenic Ia+ M phi significantly decreased (29%) during tumor growth. More important than the decreased cell numbers was the significant decrease in Ia antigen expression per cell. Unlike the peritoneal M phi, the splenic M phi did not show distinct size versus antigen expression subpopulations, although there was an overall difference in M phi size between normal and TBH. These data suggested that M phi from different anatomical sites are phenotypically different and tumor growth mediates phenotypic alterations in peritoneal and splenic M phi populations. This may be the source of tumor-induced dysfunction of M phi-mediated immune activity.

Tumor modulation of autoreactivity: Decreased macrophage and autoreactive T cell interactions☆

The autologous mixed lymphocyte reaction (AMLR) is an in vitro measure of autoreactivity, a key mechanism in immune homeostasis. In this system, macrophages (M phi) act as accessory cells to autoreactive L3T4+ T cells by presenting self-Ia and releasing soluble modulators. During tumor growth, changes occur in M phi and T cells. Tumor-bearing host (TBH) M phi have a reduced ability to act as accessory cells. In fact, TBH M phi suppressed autoreactivity by 60-70%. The decrease in TBH M phi or T-cell abilities was not due to differences in cell numbers or incubation time. Because tumor growth causes increased prostaglandin E2 (PGE2) production by M phi, indomethacin was used to assess the contribution of prostaglandins. Normal and TBH T-cell reactivity increased nearly 50% when stimulated by normal host M phi, while normal and TBH T-cell reactivity increased nearly 100% when stimulated by TBH M phi. Thus increased prostaglandin production is partly responsible for the increased TBH suppressor M phi activity and in the normal host, suppressor M phi may be responsible for maintaining immune regulation. To assess the direct role of prostaglandins in T-cell hyporesponsiveness, PGE2 was titrated into the cultures. PGE2 suppressed normal and TBH T-cell responsiveness in a dose-dependent manner. Normal host T cells were suppressed to a greater extent than TBH T cells by PGE2 (66% versus 42% suppression, respectively). Reduced Ia expression and active suppressor mechanisms are not the only mechanisms mediating hypoautoreactivity during tumor growth. TBH autoreactive L3T4+ T cells were less responsive to self-Ia; they were only 60-80% as reactive as their normal counterparts. To address whether the helper T (TH)-cell defect involved cytokines, T cells were treated with interleukin (IL)-1, IL-2, and IL-4. In all cases, the TBH T-cell response to the factors was decreased (only 60-75% as reactive as normal T cells). Because TBH M phi-mediated suppression can override the addition of IL-1, IL-2, and IL-4, indomethacin was also added with the exogenous interleukins. This coaddition significantly enhanced normal host autoreactivity above control levels while TBH autoreactivity (the combination of TBH T cells and TBH M phi) only returned to normal host unstimulated levels. Tumor growth modulates the immune response at least by (i) decreasing the accessory cell abilities of TBH M phi through decreased Ia expression and increased production of suppressive molecules such as prostaglandins; and (ii) decreasing the responsiveness to immune enhancing factors by TH cells.

Tumor-induced alteration in macrophage accessory cell activity on autoreactive T cells

SummaryUsing a tumor-model system, differences in the accessory cell capabilities on autoreactive T cells of splenic macrophages from normal and tumor-bearing hosts (TBH) were assessed in the syngeneic mixed lymphocyte reaction. Tumor development caused a drop in autoreactivity. At 0 and 7 days of tumor growth, no drop in reactivity occurred when TBH macrophages were used as accessory cells and L3T4+ autoreactive T cells from normal mice were used as responder cells. However, by day 14, there was a 32% drop in reactivity, and by day 21 only 22% of the T cell reactivity remained when TBH macrophages were used as accessory cells. Alterations in macrophage Ia antigen during tumor growth were first investigated as the potential cause of reduced autoreactivity. Before tumor growth (day 0) 59% of the splenic macrophages were found to be Ia+. Day-7 TBH macrophages showed no difference in Ia antigen expression when compared to day 0 macrophages. However, by day 14, TBH macrophages showed a 9% decrease, and by day 21 they showed a 36% decrease in the number which were Ia+. Concomitant with the decrease in the number of Ia+ cells was a decrease in the density of Ia antigen expression on day-14 and -21 TBH macrophages. In day-14 and -21 TBH macrophages, two populations were seen that were Ia+. The first had a 10%–20% decrease in Ia antigen expression per cell while the second population had a >50% drop in Ia antigen expression per cell. By titrating and mixing TBH macrophages with normal host macrophages, we assessed whether they could actively mediate suppression of autoreactive T cells. A titratable suppressive phenomenon was demonstrated using day-21 TBH macrophages. In contrast, day-7 and -14 TBH macrophages titrated with normal host macrophages had no effect on the syngeneic mixed lymphocyte reactivity. Lastly, we investigated whether the macrophage-mediated suppression was caused by increased prostaglandin secretion. Addition of indomethacin to cultures increased autoreactive T cell reactivity stimulated by normal or TBH macrophages (59% and 99% increase, respectively). Although indomethacin reduced suppression mediated by TBH macrophages, autoreactivity did not return to levels induced by untreated or indomethacin-treated cells from a normal host. Taken together, the data suggested that tumor growth modulates the function of macrophage accessory cells with autoreactive T cells in at least two ways: by decreasing Ia antigen expression and by increasing suppressor activity.

Ia- macrophages and cytokine networks contribute to tumor-induced suppression of CD4+ autoreactive T cells.

Tumor growth changes the functions and phenotypes of macrophages (M phi) and T cells. Suppression of CD4+ T cell autoresponses during tumor growth was contributed primarily by M phi. Tumor-induced alterations in the abilities of these cells to mediate autorecognition were assessed through syngeneic mixed lymphocyte reaction (SMLR) assays. Tumor-bearing host (TBH) M phi were significantly more suppressive (60-90%) than normal host (NH) M phi, and this suppression was caused partly by reduced Ia expression. TBH Ia- M phi were significantly more suppressive (50-80%) than their NH counterparts. The suppression mechanism was controlled partly by prostaglandin E2 (PGE2), because treating cultures with indomethacin and titrated NH and TBH Ia- M phi led to increased T-cell responsiveness, although responsiveness never reached levels of assays containing unseparated M phi. Blocking studies using anti-interferon-gamma (anti-IFN-gamma) monoclonal antibodies (mAb), anti-interleukin 4 (anti-IL-4) mAb, and indomethacin suggested that IFN-gamma, IL-4, and PGE2 contributed to tumor-induced M phi-mediated suppression. Our results suggested that a quantitative shift in M phi phenotype and a qualitative shift in M phi function in addition to differences in cytokine-directed accessory activities are partly responsible for tumor-induced suppression CD4+ T cell autoresponses.

Normal and tumor-bearing host macrophage responses: variability in accessory function, surface markers, and cell-cycle kinetics

Normal and tumor-bearing host (TBH) peritoneal macrophage (M phi) responses to in vitro lipopolysaccharide (LPS) treatment were measured by assessing functional and phenotypic changes. Both normal and TBH untreated M phi suppressed mixed lymphocyte reaction (MLR) reactivity at all concentrations. Normal host M phi treated with LPS for 3 h were suppressive at all concentrations. TBH M phi treated with LPS for 3 h were not suppressive in the MLR until more than 5% were added. Surprisingly, 24 h treatment of normal and TBH M phi with LPS induced cells that significantly enhanced MLR reactivity when added at 2% or 5%. These cells were not suppressive until a 20% M phi concentration was reached. LPS treatment of normal and TBH M phi changed the percentage of cells expressing the surface markers Mac-1, -2, -3, and Ia as determined by flow cytometry. Normal host peritoneal M phi treated with LPS for 3 h had decreased Mac-1 and -3 expression, but there was no change in Mac-2 or Ia. Plating for 24 h did not change the percentage of M phi expressing Mac-1, -3, or Ia but did cause an increase in Mac-2+ M phi. Treatment of normal host M phi with LPS for 24 h led to a decrease in Mac-1+ and Ia+ M phi, no change in Mac-3+ M phi, but an increase in Mac-2+ M phi. LPS treatment of TBH M phi for 3 h decreased the number of Mac-1+ M phi, but Mac-2+, -3+, or Ia+ M phi numbers did not change. Plating TBH M phi for 24 h caused a decrease in the number of Mac-1+ M phi, no change in Mac-3+ or Ia+ M phi, but an increase in Mac-2+ M phi. Treatment with LPS for 24 h led to no change in the number of Mac-1+, -3+, or Ia+ TBH M phi, but Mac-2+ M phi increased. The phenotypic and functional changes after LPS treatment led us to ask if these changes were detectable at the level of DNA and RNA. Flow cytometric analysis of acridine orange-stained M phi was used to measure DNA and RNA levels. This analysis determines M phi cell-cycle kinetics and estimates their RNA synthesis. In normal host M phi, a 3-h LPS treatment caused a decrease of cells in G0/G1 but an insignificant change in RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor growth changes the contribution of granulocyte-macrophage colony-stimulating factor during macrophage-mediated suppression of allorecognition

Tumor-bearing host (TBH) macrophages (M phi) suppress T cell alloresponses, and this study suggests granulocyte-macrophage colony-stimulating factor (GM-CSF), a molecule associated with suppressive M phi activity during tumor growth, signals more immunosuppression. In the absence of M phi, GM-CSF increased T cell proliferation in response to alloantigen. However, TBH M phi-mediated suppression of allorecogntion was further induced by GM-CSF. Allogeneic mixed lymphocyte reaction (MLR) cultures, containing normal host (NH) M phi, were either unaffected or enhanced. Prostaglandin E2 (PGE2), a highly suppressive monokine that decreases alloreactivity, did not seem to be involved in the suppression caused by the TBH M phi/GM-CSF interaction. M phi-CSF (M-CSF) addition to cultures did not reverse the suppression caused by TBH M phi and GM-CSF, and inhibition of PGE2 synthesis did not change the response to M-CSF. TBH Ia- M phi, a suppressor population that predominates among splenic M phi during tumor growth, demonstrated significantly lower reactivity in the presence of GM-CSF. In contrast, alloresponses suppressed by NH Ia- M phi demonstrated higher reactivity in the presence of GM-CSF. The data collectively suggest that TBH M phi respond differently to GM-CSF, and that tumor-induced changes in GM-CSF responsiveness affect M phi accessory ability.

Normal and Tumor-Bearing Host Splenic Macrophage Responses to Lipopolysaccharide

Lipopolysaccharide (LPS) was used to assess the responses of normal and tumor-bearing host (TBH) macrophages (M phi) to activation signals. M phi were incubated with LPS for either 3 hr or 24 hr and then assayed for phenotypic, functional, or cell-cycle changes. A 3-hr LPS treatment had no significant effect on M phi phenotype. In contrast, the 24-hr LPS treatment caused a significant decrease in the percentage of normal host Mac+ and Ia+ M phi. In the TBH, a 24-hr LPS treatment caused an increase in the percentage of Mac-1+ and -2+ M phi and a decrease in the percentage of Mac-3+ and Ia+ M phi. When normal host M phi were plated for 24 hr (control), there was an increase in Mac-1+ and -2+ M phi and a decrease in Ia+ M phi. The 24-hr TBH control showed a decrease in Mac-1+ and Ia+ M phi. To assess functional changes, LPS-treated normal and TBH M phi were added to the allogeneic mixed lymphocyte reaction (MLR) or the autologous MLR (AMLR). There were no significant differences in M phi accessory activity after a 3-hr LPS treatment. A 24-hr LPS treatment of normal host M phi also had no effect. A 24-hr LPS treatment of TBH M phi led to a significant decrease in allogeneic T-cell reactivity, and even the 24-hr TBH control showed significant suppression of T-cell responsiveness. In the AMLR, a measure of autoreactive T-cell responsiveness, a 3-hr LPS treatment had no affect on normal host M phi but led to increased accessory ability in TBH M phi. TBH M phi, however, were still less than 50% as responsive as normal host M phi even after LPS treatment. The 24-hr LPS treatment caused a significant decrease in normal or TBH M phi accessory activity. A 24-hr plating of normal host M phi decreased their accessory ability. In addition to the phenotypic and functional changes after LPS treatment, M phi cell-cycle kinetics were also investigated. The percentages of normal host M phi in G0/G1 were not changed significantly after a 3-hr LPS treatment. In contrast, M phi plated for 24 hr with or without LPS had a decreased percentage of cells in G0/G1. Normal host M phi showed little change in total RNA levels after a 3-hr treatment but had increased RNA levels after a 24-hr treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophages stimulated by receptor-ligand interactions exhibit differences in cell-cycle kinetics during tumor growth: stimulation at Mac-1 and Mac-3 receptors alters DNA synthesis.

Phenotypic and functional changes associated with tumor-bearing host (TBH) macrophages (M phi) are partly responsible for immunosuppression during tumor growth. Flow cytofluorometric analyses revealed differences in cell-cycle kinetics between normal host (NH) and TBH M phi that were stimulated at specific receptors. Receptor-ligand interactions were induced by antibodies against Mac-1, -2, -3, and Ia receptors and changes in DNA synthesis were measured over a 12-h time course by incorporation of propidium iodide. TBH M phi showed higher DNA synthesis than NH M phi over this time course irrespective of the receptor induced. NH M phi stimulated at the Mac-1 receptor demonstrated higher DNA synthesis than control NH M phi although TBH M phi stimulated at this receptor and control TBH M phi failed to show any differences. Both NH and TBH M phi exhibited small, short-term decreases in DNA synthesis when stimulated at the Mac-2 receptor. TBH M phi that were stimulated at the Mac-3 receptor demonstrated higher DNA synthesis than their control counterparts while NH M phi stimulated at this receptor and control NH M phi showed identical levels of DNA synthesis. No differences in DNA synthesis were found among normal or TBH M phi that were stimulated through Ia. Differences in DNA synthesis did not appear to be attributable to differences in receptor expression. Further analysis of Mac-1 and Mac-3 stimulated cells revealed that DNA synthesis in NH M phi stimulated at the Mac-1 receptor returned to control levels at 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)