Matthew L. Nicotra

Two Unique Human Decidual Macrophage Populations

Several important events occur at the maternal–fetal interface, including generation of maternal–fetal tolerance, remodeling of the uterine smooth muscle and its spiral arteries and glands, and placental construction. Fetal-derived extravillous trophoblasts come in direct contact with maternal decidual leukocytes. Macrophages represent ∼20% of the leukocytes at this interface. In this study, two distinct subsets of CD14+ decidual macrophages (dMɸs) are found to be present in first-trimester decidual tissue, CD11cHI and CD11cLO. Gene expression analysis by RNA microarray revealed that 379 probes were differentially expressed between these two populations. Analysis of the two subsets revealed several clusters of coregulated genes that suggest distinct functions for these subsets in tissue remodeling, growth, and development. CD11cHI dMɸs express genes associated with lipid metabolism and inflammation, whereas CD11cLO dMɸs express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The CD11cHI dMɸs also differ from CD11cLO dMɸs in their ability to process protein Ag and are likely to be the major APCs in the decidua. Moreover, these populations each secrete both proinflammatory and anti-inflammatory cytokines that may contribute to the balance that establishes fetal–maternal tolerance. Thus, they do not fit the conventional M1/M2 categorization.

HLA-G homodimer-induced cytokine secretion through HLA-G receptors on human decidual macrophages and natural killer cells

Human decidual CD14+ macrophages and CD56+ NK cells were isolated from material obtained after first-trimester pregnancy terminations. Each cell type expressed a specific surface receptor for histocompatibility leukocyte antigen (HLA)-G (an MHC class Ib protein that is expressed on extravillous trophoblasts), LILRB1 on CD14+ macrophages and KIR2DL4 on CD56+ NK cells. Cross-linking with anti-LILRB1 or anti-KIR2DL4 resulted in up-regulation of a small subset of mRNAs including those for IL-6, IL-8, and TNFα detected using a microarray representing 114 cytokines. Incubation with transfectants expressing the HLA-G homodimer (but not with transfectants expressing the HLA-G monomer) resulted in secretion of the same cytokine proteins from both leukocyte sets. Moreover, cytokine secretion from both leukocyte sets was blocked by both the appropriate anti-receptor mAb and by anti-HLA-G. The amount of these cytokines secreted by decidual macrophages was substantially greater than that secreted by decidual NK cells. VEGF was constitutively secreted by both cell types. LILRB1, which contains an immunoreceptor tyrosine-based switch motif, functions here as an activating receptor, although it has been known as an inhibitory receptor. KIR2DL4 also functions as an activating receptor, although it also has the potential to function as an inhibitory receptor. Secretion of proinflammatory and proangiogenic proteins supports a role for these leukocytes in important processes that are essential for successful pregnancy, but they may represent only a portion of the proteins that are secreted.

JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity

Interaction of the activating receptor NKG2D with its ligands is a major stimulatory pathway for cytotoxicity of natural killer (NK) cells. Here, the signaling pathway involved after NKG2D ligation is examined. Either incubation of the NKG2D-bearing human NKL tumor cell line with K562 target cells or cross-linking with NKG2D mAb induced strong activation of the mitogen-activated protein (MAP) kinases. Selective inhibition of JNK MAP kinase with four different means of inhibition greatly reduced NKG2D-mediated cytotoxicity toward target cells and furthermore, blocked the movement of the microtubule organizing center (MTOC), granzyme B (a component of cytotoxic granules), and paxillin (a scaffold protein) to the immune synapse. NKG2D-induced activation of JNK kinase was also blocked by inhibitors of Src protein tyrosine kinases and phospholipase PLCγ, upstream of JNK. Similarly, a second MAP kinase pathway through ERK was previously shown to be required for NK cell cytotoxicity. Thus, activation of two MAP kinase pathways is required for cytotoxic granule and MTOC polarization and for cytotoxicity of human NK cells when NKG2D is ligated.

A Hypervariable Invertebrate Allodeterminant

Colonial marine invertebrates, such as sponges, corals, bryozoans, and ascidians, often live in densely populated communities where they encounter other members of their species as they grow over their substratum. Such encounters typically lead to a natural histocompatibility response in which colonies either fuse to become a single, chimeric colony or reject and aggressively compete for space. These allorecognition phenomena mediate intraspecific competition, support allotypic diversity, control the level at which selection acts, and resemble allogeneic interactions in pregnancy and transplantation. Despite the ubiquity of allorecognition in colonial phyla, however, its molecular basis has not been identified beyond what is currently known about histocompatibility in vertebrates and protochordates. We positionally cloned an allorecognition gene by using inbred strains of the cnidarian, Hydractinia symbiolongicarpus, which is a model system for the study of invertebrate allorecognition. The gene identified encodes a putative transmembrane receptor expressed in all tissues capable of allorecognition that is highly polymorphic and predicts allorecognition responses in laboratory and field-derived strains. This study reveals that a previously undescribed hypervariable molecule bearing three extracellular domains with greatest sequence similarity to the immunoglobulin superfamily is an allodeterminant in a lower metazoan.

An invertebrate histocompatibility complex.

We have developed defined genetic lines of the hydroid Hydractinia symbiolongicarpus and confirmed earlier results showing that allorecognition is controlled by a single chromosomal region within these lines. In a large backcross population, we detected recombinants that display a fusibility phenotype distinct from typical fusion and rejection. We show that this transitory fusion phenotype segregates in a fashion expected of a single Mendelian trait, establishing that the chromosomal interval contains at least two genes that interact to determine fusibility. Using bulked segregant analysis, we have identified amplified fragment length polymorphisms (AFLP) cosegregating with fusibility, used these markers to independently confirm linkage of the two loci, and constructed a 3.4-cM map of an invertebrate histocompatibility complex.

Model Systems of Invertebrate Allorecognition

Nearly all colonial marine invertebrates are capable of allorecognition--the ability to distinguish between self and genetically distinct members of the same species. When two or more colonies grow into contact, they either reject each other and compete for the contested space or fuse and form a single, chimeric colony. The specificity of this response is conferred by genetic systems that restrict fusion to self and close kin. Two selective pressures, intraspecific spatial competition between whole colonies and competition between stem cells for access to the germline in fused chimeras, are thought to drive the evolution of extensive polymorphism at invertebrate allorecognition loci. After decades of study, genes controlling allorecognition have been identified in two model systems, the protochordate Botryllus schlosseri and the cnidarian Hydractinia symbiolongicarpus. In both species, allorecognition specificity is determined by highly polymorphic cell-surface molecules, encoded by the fuhc and fester genes in Botryllus, and by the alr1 and alr2 genes in Hydractinia. Here we review allorecognition phenomena in both systems, summarizing recent molecular advances, comparing and contrasting the life history traits that shape the evolution of these distinct allorecognition systems, and highlighting questions that remain open in the field.

Hydractinia allodeterminant alr1 resides in an immunoglobulin superfamily-like gene complex.

Allorecognition, the ability to discriminate between self and nonself, is ubiquitous among colonial metazoans and widespread among aclonal taxa. Genetic models for the study of allorecognition have been developed in the jawed vertebrates, invertebrate chordate Botryllus, and cnidarian Hydractinia. In Botryllus, two genes contribute to the histocompatibility response, FuHC and fester. In the cnidarian Hydractinia, one of the two known allorecognition loci, alr2, has been isolated, and a second linked locus, alr1, has been mapped to the same chromosomal region, called the allorecognition complex (ARC). Here we isolate alr1 by positional cloning and report it to encode a transmembrane receptor protein with two hypervariable extracellular regions similar to immunoglobulin (Ig)-like domains. Variation in the extracellular domain largely predicts fusibility within and between laboratory strains and wild-type isolates. alr1 was found embedded in a family of immunoglobulin superfamily (IgSF)-like genes, thus establishing that the ARC histocompatibility complex is an invertebrate IgSF-like gene complex.

Differential Effect of Allorecognition Loci on Phenotype in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa)

The allorecognition complex of Hydractinia symbiolongicarpus is a chromosomal interval containing two loci, alr1 and alr2, that controls fusion between genetically distinct colonies. Recombination between these two loci has been associated with a heterogeneous class of phenotypes called transitory fusion. A large-scale backcross was performed to generate a population of colonies (N = 106) with recombination breakpoints within the allorecognition complex. Two distinct forms of transitory fusion were correlated with reciprocal recombination products, suggesting that alr1 and alr2 contributed differentially to the allorecognition response. Specifically, type I transitory fusion is associated with rapid and persistent separation of allogeneic tissues, whereas type II transitory fusion generates a patchwork of continuously fusing and separating tissues.

A test for larval kin aggregations.

Hart and Grosberg (1) claimed that demersal larvae remain in kin aggregations. A simple test of this proposition has recently become possible. In the athecate colonial hydroid Hydractinia symbiolongicarpus, allorecognition is controlled by a single chromosomal interval and fusion is expected only between closely related individuals (2). If Hydractinia larvae do aggregate in kin groups, then recruits from the same shell should fuse more frequently than those from different shells. We sampled a shallow subtidal population from Long Island Sound (Old Quarry Harbor, Guilford, CT) to identify hermit crab shells that bore two or more newly recruited Hydractinia colonies. The colonies were explanted from the shells, reared in the laboratory, and subsequently tested for fusibility. We observed fusion between 4.4% of co-occurring colonies and 1.8% of colonies from different shells—frequencies that do not differ significantly. These results provide no support for the claim that larvae remain aggregated in the field. Hydractinia symbiolongicarpus (Buss and Yund, 1989) occurs most frequently as an epibiont on the shells of hermit crabs and is, therefore, a sessile organism on mobile substrata. Colonies are dioecious, with the gametes freely shed into the water column on a diurnal light cue. This natural history suggests that larval kin aggregations are unlikely; nonetheless, egg release is restricted to a period of less than an hour each day, the eggs are negatively buoyant, and the larvae crawl but do not swim. Thus, larvae might conceivably remain in kin aggregations if a hermit crab remained immobile; if the larvae did not crawl away from one another; and if tidal movement, currents, or bioturbation did not resuspend the mud and sand for a period of 48–72 h. Should this concatenation of conditions occur, hermit crabs might be expected occasionally to pick up two larvae as they pass through a single aggregation. In contrast, different crabs would not be likely to traverse the same aggregation; hence larvae recruiting to different shells would be expected to be unrelated. A direct test of the claim that demersal larvae remain in kin aggregations is provided by comparing new recruits found on the same shell with those on different shells. To make this test, we exploit new findings on the genetics of allorecognition in Hydractinia (2). When recruits encounter one another, one of two genetically alternative results are obtained. Colonies either fuse, establishing a chimera with a continuous gastrovascular system, or they fail to fuse (2–5). The failure to fuse is called rejection and triggers a nematocyst-based effector system, which in turn results in either the elimination of one colony or the development of the oft-observed “suture-lines” separating colonies on the same shell (6–8). We have recently demonstrated that the decision to fuse or reject is controlled by a single chromosomal interval and confirmed this result by both classical genetic techniques and the development of molecular markers spanning the interval (2). Fusion occurs only if colonies share alleles at one or more loci in this interval (2). Thus, field-collected colonies are expected to fuse only if they are related or if both carry an allele common in the population. The latter is expected to occur infrequently, as allorecognition loci are said to be highly polymorphic (9). If larvae are aggregated in kin groups, then fusion among recruits on a single shell must exceed fusion among recruits from different shells. To test Hart and Grosberg’s (1) larval kin aggregation hypothesis, we sought conditions most conducive to the formation of larval kin aggregations. Specifically, we avoided intertidal habitats and those with high currents, and we sampled from a calm, shallow subtidal embayment. Using scuba, we collected 1369 shells on four dates in 2002 (30 May, 4 June, 21 June, and 31 July) at a depth of 5 m on the seaward edge of One Tree Island, Old Quarry Harbor (Guilford, CT). Newly recruited colonies ( 10 polyps) are Received 20 July 2004; accepted 17 March 2005. * To whom correspondence should be addressed. Reference: Biol. Bull. 208: 157–158. (June 2005)

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

Detection of nonself SIRPα by CD47 triggers an innate immune response to allografts in mice that lack T, B, and NK cells. Looking beyond MHCs in transplant rejection Mice engineered to lack T, B, and NK cells generate mature dendritic cells in response to allogeneic transplants. Precisely how these mice recognize allografts to be “nonself” has remained a mystery. Using an elegant positional cloning approach, Dai et al. have identified polymorphisms in the mouse gene encoding signal regulatory protein α (SIRPα) to be key in this innate self-nonself recognition. They show that SIRPα receptor CD47 binds SIRPα variants with distinct affinities and propose this affinity sensing to be the mechanism that triggers dendritic cell maturation, the first step in the initiation of the alloimmune response. Given that the SIRPα gene is also polymorphic in humans, it remains to be seen whether human SIRPα variations influence transplantation success. Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2−/−γc−/− mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient’s innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.