Neutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca 2؉ mobilization by the CXCR2-specific chemokine, growth-related oncogene ␣ (Gro␣), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene ␣ did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca 2؉ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross-desensitization of its Ca 2؉ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca 2؉ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C ␤ 3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation.

Adolescent non-suicidal self-injury (NSSI) and suicidality are serious health concerns; however, factors that contribute to the transition from NSSI to suicide ideation and suicide attempts are unclear. To address this gap, we investigated whether demographic characteristics, child maltreatment, and psychiatric factors are associated with the level suicidality among adolescents with a history of self-injury. Participants were three groups of adolescent inpatient self-injurers (n = 397, 317 female), aged 13–18 years (M = 15.44, SD = 1.36): (a) non-ideators (n = 96; no current suicide ideation and no lifetime suicide attempts), (b) suicide ideators (n = 149; current ideation and no lifetime attempts), and (c) suicide attempters (n = 152; current ideation and at least one lifetime attempt). Participants completed interviews assessing psychiatric diagnoses, suicidality, and NSSI characteristics, as well as questionnaires on childhood trauma, psychiatric symptoms, and risky behavior engagement. Depression severity was associated with greater odds being a suicide ideator (p < 0.001, OR = 1.04) and an attempter (p < 0.001, OR = 1.05) compared to a non-ideator. Suicide attempters used more NSSI methods and reported greater risky behavior engagement than non-ideators (p = 0.03, OR = 1.29 and p = 0.03, OR = 1.06, respectively) and ideators (p = 0.015, OR = 1.25 and p = 0.04, OR = 1.05, respectively); attempters used more severe NSSI methods (e.g., burning). Our results identify a wide range of risk markers for increasing lethality in a sample at high risk for suicide mortality; future research is needed to refine risk assessments for adolescent self-injurers and determine the clinical utility of using risk markers for screening and intervention.

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B 4 (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a G␣ i inhibitor. These receptors were chosen to represent the spectrum of G i usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B 4 , and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G i , whereas BLTR and PAFR utilize both G i and a Ptxinsensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G i -mediated pathway.Migration of leukocytes to sites of inflammation is mediated via the activation of G-protein-coupled chemoattractant receptors (1, 2). Chemoattractants at low concentrations elicit shape change, pseudopod extension, and chemotaxis, and at higher doses; many of them also trigger degranulation and generation of superoxide anions (1, 3). Pathways leading to these activities have been shown to have different dose requirements, kinetics and regulation (4, 5), but the role of G-protein usage remains unknown.Formylpeptides (fMLP), 1 platelet-activating factor (PAF), and leukotriene B 4 (LTB 4 ) are potent chemoattractants for neutrophils and to varying degrees also activate exocytosis and generation of superoxide anions (3,4,6). These activities are mediated through G-protein-coupled receptors (FR, PAFR, and BLTR) (2, 7). G-protein usage of chemoattractant receptors was known to be different depending on cell types (1, 8 -10). Previous studies in RBL cells indicated that FR activated G i , whereas PAFR utilized both G i and a Ptx-insensitive G-protein to activate phosphoinositide hydrolysis, calcium mobilizatio...

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase C␤ (PLC␤) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by ϳ30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTP␥S binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca 2؉ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLC␤ 3 phosphorylation and cross-desensitization of Ca 2؉ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca 2؉ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLC␤ 3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways. IL-81 is a member of the structurally related family of cytokines, called chemokines, which mediate a number of biological activities including chemotaxis of leukocytes (1, 2). Cellular responses to IL-8 are initiated by specific cell surface receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins (3). Two IL-8 receptor subtypes have been identified in human neutrophils, IL-8 receptor A or CXCR1 and IL-8 receptor B or CXCR2 (4, 5). CXCR1 is specific for IL-8, whereas CXCR2 also binds other chemokines including MGSA, GRO, and NAP2, to mediate cellular responses (3, 6). Both CXCR1 and CXCR2 undergo phosphorylation and desensitizat...

Objective Presently, little is known about what factors predict adolescent psychiatric rehospitalization. Thus, the present study tested whether a battery of demographic and clinical characteristics predicted readmission within 6 months of discharge. Methods Participants were 165 adolescents (112 females) aged 13–19 years (mean = 15.61, SD = 1.48) admitted to an acute residential treatment program between November 25, 2013, and November 18, 2014. Patients met diagnostic criteria (DSM-IV-TR) for current major depressive disorder or dysthymia. At admission, participants completed a battery of clinical interviews and questionnaires assessing demographics, early life stress, comorbid diagnoses, psychiatric symptoms, suicidality, self-injury, and risky behavior engagement. At discharge, psychiatric symptoms were reassessed. Readmission to the same residential service was monitored over a 6-month period following discharge. Results Overall, 12.1% of adolescents were rehospitalized. We conducted a series of Cox regression survival analyses to test demographic and clinical predictors of patients’ time to readmission. More frequent self-injurious behaviors in the month prior to hospitalization was significantly associated with a more rapid time to rehospitalization (β = 0.05, SE = .02, Wald1 = 4.35, P = .037, OR = 1.05, 95% CI = 1.003–1.10). Conclusions It is critical to more effectively manage self-injury during the treatment of depressed adolescents, as this is the strongest predictor of later rehospitalization.

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1␣, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca 2؉ mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca 2؉ mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (⌬CCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca 2؉ mobilization. S2, S3, and ⌬CCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca 2؉ mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca 2؉ mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca 2؉ mobilization by S3 and ⌬CCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and ⌬CCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca 2؉ mobilization) which is inversely corrolated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.

Adolescence is a period during human development characterized by a variety of biological, psychological, and social changes. Navigating these changes can be a stressful experience for both adolescents and their families. To complicate matters further, the Internet has altered the landscape of human interaction in a way that may accentuate deficits in the capacity for self-sustaining, reciprocal peer relationships. Adolescents suffering from emotional and behavioral disorders may be especially prone to this influence, as evidenced by our observation of the growing clinical trend of adolescents admitted to inpatient and residential psychiatric units who present with a history of risky cyber-behaviors. Within these settings, education for adolescents and their families around appropriate use of the Internet, as well as social training for the online management of the impulsivity and poor judgment that is so often characteristic of adolescence, is vital. Milieu models employed in the treatment of emotionally troubled adolescents must adapt so as to incorporate the identification of problematic attachment behaviors not only in real-time relationships, but also as those behaviors inevitably occur in more troubling and potentially destructive ways over the Internet. The article addresses this need by offering recommendations for the creation of a skills-based, Internet-focused curriculum for inpatient and residential programs targeting at-risk adolescents. Evaluating the association between online communication habits and the evolution of disturbances in attachment systems is an important future direction for research aimed at safeguarding the emotional and physical well-being of all adolescents.

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