Alternatively activated macrophages recruited by Brugia malayi

The adult stage of the parasitic nematode Brugia malayi can live in the host lymphatics for many years and must have an extremely effective immuno-suppressive mechanism that prevents rejection. We have discovered that this parasite can induce alternatively activated IL-4 dependent macrophages that can block proliferation of other cells via a receptor-mediated mechanism. The proliferative block is reversible and is not a result of apoptosis. Furthermore, these suppressive cells can reduce the proliferation of a wide range of human cancer cell lines. These data demonstrates that B. malayi can lead to the activation of a novel mechanism of proliferative suppression, via IL-4 dependent macrophages. These macrophages may have important roles in altering host immune responses during parasitic infection and may even have the potential to reduce tumour cell growth. Another feature of B. malayi infection is a bias towards a type 2 immune response. To investigate the role that the B.malayi recruited antigen presenting cells have on naïve T cells, the suppressive macrophages recruited by B. malayi was used to stimulate naïve T cells from ICR transgenic mice. Although the proliferation of the naïve T cells was inhibited by parasite-induced macrophages during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, naïve T cells primed by suppressive macrophages differentiated into IL4 producing Th2 cells upon secondary stimulation instead of IFN-y producing Thi cells, as has been previously described. Th2 differentiation was associated with the inhibition of (or failure to stimulate) IFN-y producing T cells during primary stimulation. Interestingly, blocking antibodies against TGF-J3 (but not IL-b) restored the differentiation of IFN-'y producing Thi cells. These data indicate that T cells exposed to parasite induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies helminth infection. In order to identify genes that are highly expressed in suppressive macrophages, which could be involved in proliferative suppression and Th2 induction, a eDNA library was constructed from these alternatively activated macrophages and an expressed sequence tag (EST) strategy was used to characterise the abundantly expressed genes in these cells. The gene YM1/ECF-L, also recently characterised as a novel eosinophil chemotactic factor (ECF-L), was massively upregulated in this library, representing 8.5% of the EST clones. The recruitment of eosinophils is another key feature of nematode infection and indeed many other type 2 immune responses such as allergic responses. Interestingly, we found that in vivo administration of the Brugia malayi homologue of the cytokine MIF (BmMIF-l) also induced YM1/ECF-. L up-regulation. A single amino acid mutation that abolishes enzyme activity of BniMIF-1 (called BmMJF'-lG) also abolishes induction of YM1/ECF-L in F4/80 macrophages. BmMIF-1 injected mice had much higher levels of eosinophilia than BmIvllF-1G injected mice, suggesting that eosinophil recruitment is linked to the enzyme activity of BmIVIIF1. Thus, parasite derived MIF could activate macrophages to produce YM1/ECF-L, which in turn recruits eosinophils to the local site of inflammation. In order to identify IL-4 dependent genes in alternatively activated macrophages and also potentially discover novel genes involved in the mechanism of proliferative suppression, we took a number of molecular approaches. Firstly, we constructed a eDNA library from non-suppressive macrophages that are recruited in parasite implanted IL-4 deficient mice and again used an expressed sequence tag (EST) strategy to characterise the abundantly expressed genes in these cells. Comparison of the IL-4' macrophage EST dataset with the suppressive macrophage EST dataset revealed a number of potentially differentially expressed genes. Secondly, we have made a subtractive library by using 'driver' RNA from non-suppressive IL-4 deficient PEC to subtract away 'tester' RNA from suppressive macrophages, yielding a population of 20 000 recombinants enriched in putative suppression generating transcripts. A preliminary assay of 176 randomly selected clones from this library has revealed that between 10-20% of the recombinants are differentially expressed between the 2 macrophage populations. Thus, we have found that alternatively activated macrophages could play a crucial role in: 1) immune suppression, 2) inducing Th2 differentiation of naïve T cells and 3) recruiting eosinophils to the site of inflammation. We have also made the first steps towards defining the molecular basis of IL-4 dependent genes in alternatively activated macrophages, which could lead to the identification of a novel mechanism that inhibits proliferation. GLOSSARY AAM Alternatively activated macrophages APC Antigen presenting cell BALF Bronchoalveolar lavage fluid CAMO Classically activated macrophages CFSE Carboxyfluorescein diacetate succinimidyl ester CTL Cytotoxic T lymphocytes EAIE Experimental autoimmune encephalomyelitis ELISA Enzyme-linked immunosorbent assay EST Expressed sequence tags F1TC Fluorescein isothiocyanate i.p. Intraperitoneal IFN Interferon IL Interleukin LPS Lipopolysaccharide Mf Microfilaria M Macrophages MHC Major histocompatibility complex MW Macrophage migration inhibitory factor NO Nitric Oxide PE Phycoerythrin PEC Peritoneal exudate cells TCR T cell receptor TGF Transforming growth factor Th T helper cell WT Wild type