Allorecognition Triggers Autophagy and Subsequent Necrosis in the Cnidarian <em>Hydractinia symbiolongicarpus</em>

<div><p>Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid <em>Hydractinia symbiolongicarpus</em> when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous <em>in vivo</em> digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion <em>in vivo</em> in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.</p> </div

Transmission electron micrographs of allogeneic colony fusion.

<p>(A) Ectodermal margin of separated <i>fr/ff</i> versus <i>rr/rr</i> fusion zone after separation (3,000X). Arrows to glycocalyx, double-headed arrows to points of apparent loss of membrane integrity. (B) Contact zone between <i>rf/ff</i> versus <i>rr/rr</i> encounter (3,000X). Arrows point to multivestivular bodies (MVB), double-headed arrow designates point of contact. (C) MVB (12,000X). (D) Multilamellar body (MLB) (20,000X). Gastroderm of (E) <i>rf/ff</i> versus <i>rr/rr</i> encounter (20,000X) and of (F) <i>rf/ff</i> versus <i>rr/rr</i> (12,000X), showing numerous phagophores and autophagosomes (arrows). MVB, multivesticular body; N, nucleus; NC, necrotic cell; Ph, phagophores; T, tear in section.</p

(A) Allogeneic colony fusion 22 hours post-contact (42X).

<p>(B) Same encounter as (A) at 55 hours post-contact. Arrowheads point to separation. (C) Scanning electron micrograph of colonies undergoing transitory fusion (80X). (D) Allogeneic polyp fusion showing necrotic cells shed at the graft boundary (108X).</p

Transmission electron micrographs of allogeneic polyp fusions.

<p>(A) Ectoderm at graft margin (4400X) 24 hours post-fusion, arrows to multivesticular bodies, double-shafted arrows to sites of apparent loss of membrane integrity. (B) Ectodermal surface at graft margin 24 hours post-fusion showing vacuolar ghosts (20000X) including (C) mitochondria remnants (85000X). (D) Ectoderm at graft margin at 48 hours post-fusion (12000X). (E) Ectoderm at graft margin at 48 hours post-fusion (20000X). MLB, multilamellar body; N, nucleus; NC, necrotic cell.</p

Transmission electron micrographs of isogeneic fusions.

<p>(A) Colony assay (3000X). (B) Polyp assay (4400X). Arrows point to mesoglea, BEc, basal ectoderm; Ec, ectoderm; En, endoderm; GVC, gastrovascular cavity, N, nucleus.</p

Phenotype, function, and differentiation potential of human monocyte subsets

<div><p>Human monocytes have been grouped into classical (CD14⁺⁺CD16⁻), non-classical (CD14^dimCD16⁺⁺), and intermediate (CD14⁺⁺CD16⁺) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1β in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.</p></div