The interaction statistics for the normalized data (Table 4, right, Interaction Effect) reveal that only IL10 and CXCL1 are significantly different with BMDMs having the greater response (i

The interaction statistics for the normalized data (Table 4, right, Interaction Effect) reveal that only IL10 and CXCL1 are significantly different with BMDMs having the greater response (i.e., fold change, bold text). Each symbol represents cells from one mouse. * 0.05, ** PDE12-IN-3 0.005 compared to no treatment. studies. Given that pMACs mature while BMDM are differentiated from stem cells, it is likely that their responses differ under experimental PDE12-IN-3 conditions. Surprisingly little is known about how BMDM and pMACs responses compare under the same experimental conditionals. While morphologically similar with respect to forward and side scatter by flow cytometry, reports in the literature suggest that pMACs are more mature than their BMDM counterparts. Given the dearth of information comparing BMDM and pMACs, this work was undertaken to test the hypothesis that elicited pMACs are more responsive to defined conditions, including phagocytosis, respiratory burst, polarization, and cytokine and chemokine release. In all cases, our hypothesis was disproved. At steady state, BMDM are more phagocytic (both rate and extent) than elicited pMACs. In response to polarization, they upregulate chemokine and cytokine gene expression and release more cytokines. The results demonstrate that BMDM are generally more responsive and poised to respond to their environment, while pMAC responses are, in comparison, less pronounced. BMDM responses are a function of intrinsic differences, while pMAC responses reflect their differentiation in the context of the whole animal. This distinction may be important in knockout animals, where the pMAC phenotype may be influenced by the absence of the gene of interest. and reported differences between the two (3, 6, 7), it is somewhat surprising that the two have not been compared with respect to the properties that define macrophages: phagocytosis, respiratory burst, polarization, and gene regulation. Despite reports that pMACs are more mature (and thus respond more robustly to stimulation), we found that BMDMs are more phagocytic (rate and amount of material ingested) Rabbit polyclonal to Src.This gene is highly similar to the v-src gene of Rous sarcoma virus.This proto-oncogene may play a role in the regulation of embryonic development and cell growth.The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase.Mutations in this gene could be involved in the malignant progression of colon cancer.Two transcript variants encoding the same protein have been found for this gene. and respond more robustly to polarization (surface molecule expression, gene induction/repression, and cytokine/chemokine release). These findings are consistent with the differential PDE12-IN-3 plasticity of pMACs and BMDMs. That is, pMACs, being differentiated while BMDMs are poised to respond rapidly and robustly to either pro-inflammatory or pro-resolving stimuli = 8) CD11b+F4/80+ (Figure 1A); there is no detectable SiglecF or Ly6G. Based on forward and side scatter, BMDM have a minor population (15.8 3.4%, = 10) of large cells. As reported previously for pMACs (1), CD11b and F4/80 expression is significantly higher on large vs. small BMDM ( 0.01, = 10) (Figure 1B). Open in a separate window Figure 1 Bone marrow-derived macrophages PDE12-IN-3 exist as two distinct populations. Bone marrow was extracted and differentiated in L cell media as described in Methods. Adherent cells were collected 7 days post-harvesting and analyzed by flow cytometry (representative of BMDMs from 10 animals). (A) Virtually all (98 2%) of the live singlets were CD11b+F4/80+. (B) After gating out dead cells/debris and selecting for singlets, two populations were identified: a minor (15.8 3.4%) PDE12-IN-3 population of high forward and side scatter (large) cells and a major population that is smaller with lower side scatter. The large population had significantly higher expression of both F4/80 and CD11b ( 0.01, = 10, paired = 15). Flow cytometry revealed a low forward scatter, moderate side scatter population in the harvested pMACs (11 4.4%, = 10) that was significantly diminished upon adhesion (2.1 1.1%, = 10, 0.01, paired being CD11b+Ly6G+Ly6Clo/neg (= 10). The majority of recovered peritoneal cells (82.7 6.2 %, = 10) are CD11b+; this percentage rose significantly (91.5 2.5 %, 0.005, = 10) following adhesion (Figures 2B,C). Like BMDMs, selected pMACs contain large (~20%) and small macrophages (Figures 2A,D) (1); adhesion does not affect the relative percentages of these populations. When compared, adherent pMACs and BMDMs are similar with respect to size and granularity (Figure 2D, overlay). Open in a separate window Figure 2 The.