We identify the alpha4 subunit of integrin as a predominant integrin expressed by neural crest cells in both avian and murine embryos. Using degenerate primers, we obtained a PCR fragment of the chick integrin alpha4 subunit that was subsequently used to clone the full-length subunit with a predicted amino acid sequence 60% identical to human and mouse alpha4 subunits. In situ hybridization demonstrates that chick integrin alpha4 mRNA is expressed at high levels by migrating neural crest cells and neural crest-derived ganglia at both cranial and trunk levels. An antibody against the murine alpha4 subunit revealed similar distribution patterns in mouse to chick. In addition to neural crest cells, the integrin alpha4 subunit was later observed on the muscle masses of the limb, the apical ectodermal ridge, and the developing liver. To examine the functional role of the integrin alpha4 subunit in neural crest cell migration, we used an explant preparation that allows visualization of neural crest cells in their normal environment with or without perturbing reagents. In the presence of a blocking antibody against the mouse integrin alpha4 subunit, there was a profound abrogation of neural crest cell migration at trunk and hindbrain levels. Both the numbers of migrating neural crest cells and the total distance traversed were markedly reduced. Similarly, avian embryos injected with synthetic peptides that contain the integrin alpha4 binding site in fibronectin displayed abnormal neural crest cell migration. Our results suggest that the integrin alpha4 subunit is important for normal neural crest cell migration and may be one of the primary alpha subunits used for neural crest cell migration in vivo. Furthermore, the integrin alpha4 subunit represents a useful neural crest marker in the mouse.
Cutaneous T-cell lymphoma (CTCL) develops from clonally expanded CD4 T cells in a background of chronic inflammation. Although dendritic cells (DCs) stimulate T cells and are present in skin, cutaneous T cells in CTCL do not respond with effective antitumor immunity. We evaluated primary T-cell and DC émigrés from epidermal and dermal explant cultures of skin biopsies from CTCL patients ( = 37) and healthy donors ( = 5). Compared with healthy skin, CD4 CTCL populations contained more T cells expressing PD-1, CTLA-4, and LAG-3. CD8 CTCL populations contained more T cells expressing CTLA-4 and LAG-3. CTCL populations also contained more T cells expressing the inducible T-cell costimulator (ICOS), a marker of T-cell activation. DC émigrés from healthy or CTCL skin biopsies expressed PD-L1, indicating that maturation during migration resulted in PD-L1 expression irrespective of disease. Most T cells did not express PD-L1. Using skin samples from 49 additional CTCL patients for an unsupervised analysis of genome-wide mRNA expression profiles corroborated that advanced T3/T4-stage samples expressed more checkpoint inhibition mRNA compared with T1/T2 stage patients or healthy controls. Exhaustion of activated T cells is therefore a hallmark of both CD4 and CD8 T cells isolated from the lesional skin of patients with CTCL, with increasing expression as the disease progresses. These results justify identification of antigens driving T-cell exhaustion and the evaluation of immune checkpoint inhibition to reverse T-cell exhaustion earlier in the treatment of CTCL. .
Although it is well-established that beta1 integrins play a functional role in the migration of cranial neural crest cells, little is known about the number or importance of their associated alpha subunits. Here, we have utilized antisense oligonucleotides (aONs) against various mammalian integrin alpha subunits to functionally "knock out" integrins in vitro and in vivo. First, we examined the attachment in vitro of cranial neural crest cells to fibronectin and laminin in the presence of antisense or reversed-sense oligonucleotides using a quantitative adhesion assay. We found three alpha integrin aONs that blocked attachment to fibronectin substrates only, one that blocked attachment to laminin substrates only, and one that blocked attachment to both fibronectin and laminin. As expected, an aON to chick beta1 integrin reduced attachment to both fibronectin and laminin substrates. These results suggest that there are three or more functionally distinct integrin heterodimers on avian cranial neural crest cells. Second, we examined the ability of aONs against various alpha integrin subunits to perturb cranial neural crest migration in vivo by injecting the oligonucleotides into the cranial mesenchyme through which neural crest cells migrate. Those alpha aONs that inhibited cell attachment in vitro also caused neural crest and/or neural tube abnormalities after injection in vivo. In addition, two aONs that had no effect in vitro did affect emigration of neural crest cells in vivo. Immunoprecipitations revealed that some integrin subunits were depleted after treatment with antisense but not reversed-sense oligonucleotides both in vivo and in vitro. The results suggest that integrin alpha subunits are required for cranial neural crest cell attachment and emigration.
Cochlear inflammatory diseases such as tympanogenic labyrinthitis are associated with acquired sensorineural hearing loss. Although otitis media is extremely frequent in children, tympanogenic labyrinthitis is not commonly observed, which suggests the existence of a potent anti-inflammatory mechanism modulating cochlear inflammation. In this study, we aim to determine the molecular mechanism involved in cochlear protection from inflammation-mediated tissue damage, focusing on interleukin-10 (IL-10) and hemoxygenase-1 (HMOX1) signaling. We demonstrated that IL-10 receptors (IL-10Rs) are expressed in the cochlear lateral wall of mice and rats, particularly in the spiral ligament fibrocytes (SLFs). The rat SLF cell line (RSL) was found to inhibit nontypeable H. influenzae (NTHi)-induced up-regulation of monocyte chemotactic protein-1 (MCP-1/CCL2) in response to IL-10. This inhibition was suppressed by silencing IL-10R1 and was mimicked by cobalt protoporphyrin IX (CoPP) and carbon monoxide-releasing molecule-2 (CORM-2). In addition, IL-10 appeared to suppress monocyte recruitment through reduction of NTHi-induced RSL-derived chemoattractants. Silencing of HMOX1 was found to attenuate the inhibitory effect of IL-10 on NTHi-induced MCP-1/CCL2 up-regulation. Chromatin immunoprecipitation (ChIP) assays showed that IL-10 inhibits NTHi-induced binding of p65 NF-κB to the distal motif in the promoter region of MCP-1/CCL2, resulting in suppression of NTHi-induced NF-κB activation. Furthermore, IL-10 deficiency appeared to significantly affect cochlear inflammation induced by intratympanic injections of NTHi. Taken together, our results suggest that IL-10/HMOX1 signaling is involved in modulation of cochlear inflammation through inhibition of MCP-1/CCL2 regulation in SLFs, implying therapeutic potential of a carbon monoxide (CO)-based approach for inflammation-associated cochlear diseases.
Integrins are cell surface receptors for a variety of extracellular matrix molecules including fibronectin, laminin and collagens. Although their role in development is not completely understood, they are likely to have important functions in cell migration and axon guidance. To characterize the types of integrins expressed in the developing nervous system, we have used monoclonal antibodies against alpha 7- and alpha v-integrin subunits to examine the distribution of these subunits in the early chick embryo. Low levels of alpha 7 immunoreactivity were first observed in the neural tube and developing myotome of stage 17 embryos (E2.5). Although low levels of alpha 7 expression were associated with most neuroepithelial cells, distinct alpha 7 immunoreactivity was first detected in the ventrolateral portions of the neural tube at a stage corresponding to the time when the first neurons differentiate. Its distribution pattern overlapped with that of commissural neurons in the developing spinal cord. alpha 7 was also prominently localized to the motor neurons and their axons emanating from the neural tube. In addition, alpha 7 immunoreactivity was observed on a subpopulation of trunk neural crest cells migrating through the somitic sclerotome. At later stages, alpha 7 expression was observed in other nervous system structures such as the pigmented retinal epithelial cells. In addition to its distribution in the developing nervous system, alpha 7 immunoreactivity was associated with early myotomal cells shortly after myotome formation and its expression persisted throughout myotome development. In contrast to alpha 7, alpha v-integrin had a limited distribution in the nervous system, being expressed only at low levels in the neural tube. However, alpha v displayed prominent immunoreactivity in the myotome and in endothelial cells of the dorsal aorta. The results suggest that alpha 7-integrin is one of the prevalent integrin subunits on neurons and axons in the developing spinal cord.
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