rapid-magnetic-microbead-method-for-efficient-purification-low

Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has ...

Universidad Nacional Autónoma de México

R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype.

R-Ras promotes tumor growth of cervical epithelial cells.

R-Ras est 55 % identique &agrave; H-Ras. Cependant, ces deux oncog&egrave;nes semblent ont le potentiel de transformer la tumeur diff&eacute;rent. R-Ras induits par la transformation des cellules fibroblastes, mais pas dans les autres types de cellules. R-Ras aurait &eacute;galement induit un ph&eacute;notype plus envahissant dans les cellules &eacute;pith&eacute;liales mammaires gr&acirc;ce &agrave; l&#39;activation de l&#39;int&eacute;grine. Les auteurs ont &eacute;tudi&eacute; les m&eacute;canismes par lequel R-Ras induit un ph&eacute;notype malin.

R-Ras favorise la croissance de la tumeur des cellules &eacute;pith&eacute;liales du col ut&eacute;rin.

R-Ras &egrave; 55% identico a H-Ras. Tuttavia, questi due oncogeni sembrano avere diverso potenziale di trasformazione tumorale. R-Ras indotta trasformazione cellulare in fibroblasti, ma non in altri tipi di cellule. R-Ras induce anche riferito un fenotipo pi&ugrave; invasivo delle cellule epiteliali del seno attraverso l&#39;attivazione di integrina. Gli autori hanno studiato i meccanismi per cui R-Ras induce un fenotipo maligno.

R-Ras promuove la crescita del tumore di cellule epiteliali cervicali.

R-Ras es id&eacute;ntico al H-Ras de 55%. Sin embargo, estos dos oncogenes parecen tener diferentes posibilidades de transformar de tumor. R-Ras inducida por transformaci&oacute;n celular en fibroblastos pero no en otros tipos celulares. R-Ras tambi&eacute;n induce un fenotipo m&aacute;s invasivo en c&eacute;lulas epiteliales de mama a trav&eacute;s de la activaci&oacute;n de la integrina. Los autores estudiaron los mecanismos por el que R-Ras induce un fenotipo maligno.

R-Ras promueve el crecimiento del tumor de c&eacute;lulas epiteliales cervicales.

Mutations in the small GTPase R-Ras that promote constitutive activation of this signaling molecule have been observed in a variety of invasive cancer cell types. We previously reported that expression of an oncogenic form of R-Ras (R-Ras87L) in a cell line of cervical cancer (C33A cells) augments cell growth in vitro and tumorigenicity in vivo. Because increased tumorigenicity in vivo often precedes metastasis, we now examined whether the expression of R-Ras87L also increased the metastatic potential of C33A cells. Accelerated tumor growth was observed in athymic mice after subcutaneous injection of R-Ras87L-expressing C33A cells. In addition, increased metastasis to the liver, in immunodeficient SCID mice, was observed after intravenous injection of R-Ras87L-expressing C33A cells. Also, R-Ras87L-expressing cells presented decreased membrane expression of MHC class I molecules, and beta1 integrins, but increased levels of PI 3-K and Akt activities. C33A cells expressing R-Ras87L also migrated more over collagen I in wound assays. Inhibition of the PI 3-K/Akt/mTOR pathway by pharmacological means blocked R-Ras87L-induced accelerated growth and migration over collagen I. These results suggest oncogenic R-Ras has a central role in cancer progression towards a metastatic phenotype, through the activation of the PI 3-K/Akt/mTOR signaling pathway.

R-Ras promotes metastasis of cervical cancer epithelial cells.

Mutations dans la petite GTPase R-Ras qui favorisent l&#39;activation constitutive de cette mol&eacute;cule de signalisation ont &eacute;t&eacute; observ&eacute;es dans une vari&eacute;t&eacute; de types de cellules de cancer invasif. Nous avons d&eacute;j&agrave; indiqu&eacute; que l&#39;expression d&#39;une forme oncog&eacute;nique de R-Ras (R-Ras87L) dans une lign&eacute;e cellulaire de cancer du col ut&eacute;rin (cellules C33A) augmente la croissance cellulaire in vitro et in vivo de tumorig&eacute;nicit&eacute;. Parce qu&#39;a augment&eacute; la tumorig&eacute;nicit&eacute; in vivo pr&eacute;c&egrave;de souvent des m&eacute;tastases, nous avons examin&eacute; maintenant si l&#39;expression de R-Ras87L a aussi augment&eacute; le potentiel m&eacute;tastatique des cellules C33A. La croissance tumorale acc&eacute;l&eacute;r&eacute;e a &eacute;t&eacute; observ&eacute;e chez les souris athymiques apr&egrave;s injection sous-cutan&eacute;e de cellules C33A exprimant le R-Ras87L. En outre, accrue des m&eacute;tastases au foie, &agrave; des souris immunod&eacute;ficientes SCID, a &eacute;t&eacute; observ&eacute;e apr&egrave;s injection intraveineuse de cellules C33A exprimant le R-Ras87L. En outre, les cellules exprimant le R-Ras87L pr&eacute;sent&eacute;s une diminution membrane expression de MHC classe I mol&eacute;cules et les int&eacute;grines beta1, mais des niveaux accrus de PI 3-K et les activit&eacute;s de l&#39;Akt. Cellules C33A exprimant R-Ras87L &eacute;galement migr&eacute; plus au collag&egrave;ne je lors d&#39;essais de la plaie. Inhibition de la voie 3-K/Akt/mTOR de PI par des moyens pharmacologiques bloqu&eacute;s induite par R-Ras87L s&#39;acc&eacute;l&eacute;rant la croissance et la migration au collag&egrave;ne I. Ces r&eacute;sultats sugg&egrave;rent le qu&#39;oncog&egrave;ne R-Ras a un r&ocirc;le central dans la progression du cancer vers un ph&eacute;notype m&eacute;tastatique, par le biais de l&#39;activation de la voie de signalisation PI 3-K/Akt/mTOR.

R-Ras favorise la m&eacute;tastase des cellules &eacute;pith&eacute;liales cancer du col ut&eacute;rin.

Sono state osservate mutazioni nel piccolo GTPase R-Ras che promuovono l&#39;attivazione costitutiva di questa molecola di segnalazione in una variet&agrave; di tipi di cellule di cancro invasivo. Abbiamo segnalato in precedenza quell&#39;espressione di una forma oncogenica di R-Ras (R-Ras87L) in una linea cellulare di cancro cervicale (cellule C33A) potenziati crescita cellulare in vitro e in vivo tumorigenicity. Perch&eacute; aumentato tumorigenicity in vivo spesso precede metastasi, abbiamo ora esaminato se l&#39;espressione di R-Ras87L anche aumentato il potenziale metastatico delle cellule C33A. Crescita accelerata del tumore &egrave; stata osservata nel topo atimico dopo iniezione sottocutanea di R-Ras87L-esprimendo C33A cellule. Inoltre, &egrave; stato osservato aumento metastasi al fegato, in topi SCID immunodeficienti, dopo un&#39;iniezione endovenosa di cellule di R-Ras87L-esprimendo C33A. Inoltre, le cellule di R-Ras87L-esprimendo presentate membrana diminuita espressione di MHC classe I, molecole e le integrine beta1, ma aumento dei livelli di attivit&agrave; di Akt e PI 3K. C33A cellule che esprimono anche R-Ras87L migrato pi&ugrave; sul collagene I dosaggi di ferita. Inibizione del percorso PI 3-K/Akt/mTOR con mezzi farmacologici bloccati R-Ras87L-induced accelerata crescita e migrazione sul collagene I. Questi risultati suggeriscono che oncogenica R-Ras ha un ruolo centrale nella progressione del cancro verso un fenotipo metastatico, attraverso l&#39;attivazione del percorso PI 3-K/Akt/mTOR segnalazione.

R-Ras promuove la metastasi delle cellule epiteliali del cancro cervicale.

Se han observado mutaciones en la peque&ntilde;a GTPasa R-Ras que promueven la activaci&oacute;n constitutiva de esta mol&eacute;cula de se&ntilde;alizaci&oacute;n en una variedad de tipos de c&eacute;lulas de c&aacute;ncer invasor. Divulgamos previamente que la expresi&oacute;n de una forma oncog&eacute;nica de R-Ras (R-Ras87L) en una l&iacute;nea celular de c&aacute;ncer de cuello uterino (c&eacute;lulas C33A) aumenta crecimiento de c&eacute;lulas in vitro y in vivo de la colonizaci&oacute;n. Porque aument&oacute; la colonizaci&oacute;n in vivo a menudo precede met&aacute;stasis, ahora examinamos si la expresi&oacute;n de R-Ras87L tambi&eacute;n aument&oacute; el potencial metast&aacute;sico de c&eacute;lulas C33A. Crecimiento acelerado del tumor fue observado en ratones athymic despu&eacute;s de la inyecci&oacute;n subcut&aacute;nea de c&eacute;lulas C33A R-Ras87L-expresi&oacute;n. Adem&aacute;s, se observ&oacute; aumentada met&aacute;stasis al h&iacute;gado en ratones de SCID inmunodeficientes, despu&eacute;s de la inyecci&oacute;n intravenosa de c&eacute;lulas C33A R-Ras87L-expresi&oacute;n. Adem&aacute;s, c&eacute;lulas de R-Ras87L-expresi&oacute;n presentadas la expresi&oacute;n disminuida de la membrana de MHC clase I mol&eacute;culas y las integrinas beta1, pero los niveles crecientes de PI 3-K y actividades de Akt. C33A las c&eacute;lulas que expresan R-Ras87L tambi&eacute;n emigraron m&aacute;s sobre col&aacute;geno I en ensayos de la herida. Inhibici&oacute;n de la v&iacute;a de 3-K/Akt/mTOR de PI por medios farmacol&oacute;gicos bloqueados R-Ras87L-induced acelerado crecimiento y la migraci&oacute;n sobre el col&aacute;geno I. Estos resultados sugieren que r-Ras oncog&eacute;nica tiene un papel central en la progresi&oacute;n del c&aacute;ncer hacia un fenotipo metast&aacute;sico, mediante la activaci&oacute;n de la v&iacute;a de se&ntilde;alizaci&oacute;n PI 3-K/Akt/mTOR.

R-Ras promueve la met&aacute;stasis de las c&eacute;lulas epiteliales del c&aacute;ncer de cuello uterino.

Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

FcgammaRIIA and FcgammaRIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils.

R&eacute;cepteurs aux IgG Abs (Fcgamma r&eacute;cepteurs) sont capables de d&eacute;clencher les r&eacute;actions de diverses cellules dans les leucocytes. Neutrophiles, deux r&eacute;cepteurs de Fcgamma, &agrave; savoir FcgammaRIIA et FcgammaRIIIB, sont constitutivement exprim&eacute;s. Les voies de signalisation qui r&eacute;gulent la phagocytose m&eacute;di&eacute;e par FcgammaRIIA ont &eacute;t&eacute; relativement bien d&eacute;crites. Toutefois, les diff&eacute;rentes voies de signalisation qui m&egrave;nent &agrave; l&#39;activation de NF apr&egrave;s l&#39;engagement de chaque r&eacute;cepteur Fcgamma ont seulement &eacute;t&eacute; partiellement d&eacute;crits. Pour r&eacute;soudre ce probl&egrave;me, les neutrophiles ont &eacute;t&eacute; stimul&eacute;es par r&eacute;ticulation s&eacute;lectivement chaque type de r&eacute;cepteur de Fcgamma avec le mAbs sp&eacute;cifiques, et l&#39;activation de NF puis analys&eacute;e. FcgammaRIIIB, mais pas FcgammaRIIA, favoris&eacute; une hausse robuste de ERK phosphoryl&eacute;e dans le noyau et aussi efficace phosphorylation de la NF Elk-1. MAb complet 3 8 (anti-FcgammaRIIIB) a induit une r&eacute;ponse plus &eacute;lev&eacute;e que ne F(ab&#39;)(2) fragments de mAb 3 8, ce qui sugg&egrave;re un possible effet synergique des deux r&eacute;cepteurs de FcgammaR. Cependant, mAb IV.3 (anti-FcgammaRIIA) seul n&#39;a pas caus&eacute; une augmentation de ERK phosphoryl&eacute;e dans le noyau. Phosphorylation nucl&eacute;aire induite par le FcgammaRIIIB de ERK et de Elk-1, n&#39;a pas &eacute;t&eacute; affect&eacute;e par les inhibiteurs de Syk, PI3K ou MEK. En revanche, FcgammaRIIA - ou FcgammaRIIIB-m&eacute;diation de la phosphorylation de ERK cytoplasmique d&eacute;pendait de Syk, PI3K et MEK. ERK, mais pas de MEK, est constitutivement pr&eacute;sente dans le noyau et la r&eacute;ticulation de FcgammaRIIIB n&#39;a pas augment&eacute; les niveaux de nucl&eacute;aire ERK ou MEK. Ces donn&eacute;es montrent clairement que les diff&eacute;rents r&eacute;cepteurs de Fcgamma neutrophiles poss&egrave;dent des capacit&eacute;s diff&eacute;rentes de signalisation. FcgammaRIIIB, mais pas FcgammaRIIA, active une unique voie de signalisation conduisant &agrave; la phosphorylation de restriction nucl&eacute;aire de ERK et Elk-1, ind&eacute;pendamment de Syk, PI3K ou MEK.

FcgammaRIIA et FcgammaRIIIB m&eacute;dient activation du facteur nucl&eacute;aire par le biais de voies de signalisation distinctes dans les neutrophiles humains.

Recettori per IgG Abs (recettori Fcgamma) sono in grado di attivare risposte diverse cellule nei leucociti. In neutrofili, due recettori Fcgamma, vale a dire FcgammaRIIA e FcgammaRIIIB, sono espressi costitutivamente. Le vie di segnalazione che regolano la fagocitosi mediata da FcgammaRIIA sono state descritte relativamente bene. Tuttavia, le vie di segnalazione differenti che portano all&#39;attivazione di NF dopo il fidanzamento di ogni recettore Fcgamma solo sono stati parzialmente descritti. Per risolvere questo problema, i neutrofili sono stati stimolati dal cross-linking selettivamente ogni tipo di recettore Fcgamma con specifiche mAbs, e attivazione di NF &egrave; stato poi analizzato. FcgammaRIIIB, ma non FcgammaRIIA, promosso un robusto aumento di ERK fosforilato nel nucleo e anche efficiente fosforilazione della NF Elk-1. MAb completo 3 8 (anti-FcgammaRIIIB) ha indotto una risposta superiore rispetto a fatto F(ab&#39;)(2) frammenti di mAb 3 8, suggerendo un possibile effetto sinergico di entrambi i recettori FcgammaR. Tuttavia, mAb IV (anti-FcgammaRIIA) da solo non ha causato un aumento di ERK fosforilato nel nucleo. FcgammaRIIIB-induced nuclear fosforilazione di ERK ed Elk-1, non &egrave; stata influenzata da inibitori Syk, PI3K o MEK. Al contrario, FcgammaRIIA - o FcgammaRIIIB-mediato la fosforilazione di ERK citoplasmatica dipendeva Syk, PI3K e MEK. Inoltre, ERK, ma non MEK, era costitutivamente presente nel nucleo, e FcgammaRIIIB cross-linking non aumenta i livelli di nucleare ERK o MEK. Questi dati mostrano chiaramente che diversi recettori Fcgamma neutrofili possiedono diverse funzionalit&agrave; di segnalazione. FcgammaRIIIB, ma non FcgammaRIIA, attiva un&#39;unica via di segnalazione che porta la fosforilazione di ERK ed Elk-1, indipendentemente dalla Syk, PI3K o MEK nucleare limitata.

FcgammaRIIA e FcgammaRIIIB mediano l&#39;attivazione del fattore nucleare attraverso vie di segnalazione separate in neutrofili umani.

Receptores de IgG Abs (receptores de Fcgamma) son capaces de desencadenar respuestas de diversas c&eacute;lulas en leucocitos. En neutr&oacute;filos, dos receptores de Fcgamma, es decir, FcgammaRIIA y FcgammaRIIIB, se expresan constitutivamente. Las v&iacute;as de se&ntilde;alizaci&oacute;n que regulan la fagocitosis mediada por FcgammaRIIA se han descrito relativamente bien. Sin embargo, las diferentes v&iacute;as de se&ntilde;alizaci&oacute;n que conducen a la activaci&oacute;n de NF tras el compromiso de cada receptor de Fcgamma han s&oacute;lo parcialmente descritas. Para solucionar este problema, neutr&oacute;filos fueron estimulados por Cross-linking selectivamente cada tipo de receptor de Fcgamma con mAbs espec&iacute;ficos, y luego se analiz&oacute; la activaci&oacute;n de NF. FcgammaRIIIB, pero no FcgammaRIIA, promovi&oacute; un s&oacute;lido aumento en ERK fosforilado en el n&uacute;cleo y tambi&eacute;n eficiente fosforilaci&oacute;n de NF Elk-1. MAb completa 3 8 (anti-FcgammaRIIIB) indujo una respuesta mayor que hicieron F(ab&#39;)(2) fragmentos de mAb 3 8, sugiriendo un posible efecto sin&eacute;rgico de ambos receptores FcgammaR. Sin embargo, mAb IV.3 (anti-FcgammaRIIA) solo no provocaron un aumento de ERK fosforilado en el n&uacute;cleo. Inducida por el FcgammaRIIIB nuclear fosforilaci&oacute;n de ERK y Elk-1, no fue afectada por inhibidores Syk, PI3K o MEK. En cambio, la fosforilaci&oacute;n mediada por la FcgammaRIIA o la FcgammaRIIIB de ERK citopl&aacute;smico depend&iacute;a de Syk, PI3K y MEK. Adem&aacute;s, ERK, pero no MEK, era constitutivamente presentes en el n&uacute;cleo y FcgammaRIIIB Cross-linking no aument&oacute; los niveles de ERK nuclear o de MEK. Estos datos muestran claramente que diversos receptores de Fcgamma neutr&oacute;filos poseen diferentes capacidades de se&ntilde;alizaci&oacute;n. FcgammaRIIIB, pero no FcgammaRIIA, se activa una v&iacute;a de se&ntilde;alizaci&oacute;n &uacute;nica que conduce a la restricci&oacute;n nuclear fosforilaci&oacute;n de ERK y Elk-1, independientemente de Syk, PI3K o MEK.

FcgammaRIIA y FcgammaRIIIB median la activaci&oacute;n del factor nuclear a trav&eacute;s de diferentes v&iacute;as de se&ntilde;alizaci&oacute;n en neutr&oacute;filos humanos.

Specific receptors for antibody, named Fc Receptors are proteins found on the surface of leukocytes. Fc receptors contribute to the protective functions of the immune system, by binding to antibodies that are attached to infected cells or invading pathogens. Fc receptor activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. This review describes the main types of Fc receptors and the signal transduction pathways that are initiated by them in various leukocytes, with emphasis on activation of phagocytosis. New findings on the regulatory role of Fc receptors on immune function are also discussed.

[Fc receptor functions in host and immune regulation].

Des r&eacute;cepteurs sp&eacute;cifiques pour les anticorps, nomm&eacute; les r&eacute;cepteurs Fc sont des prot&eacute;ines pr&eacute;sentes &agrave; la surface des leucocytes. Les r&eacute;cepteurs FC contribuent aux fonctions de protection du syst&egrave;me immunitaire, par liaison aux anticorps attach&eacute;s &agrave; des cellules infect&eacute;es ou envahir les agents pathog&egrave;nes. Activit&eacute; de r&eacute;cepteur FC stimule les cellules phagocytaires ou cytotoxiques pour d&eacute;truire les microbes, ou infection de cellules par phagocytose m&eacute;di&eacute;e par les anticorps ou la cytotoxicit&eacute; &agrave; m&eacute;diation cellulaire d&eacute;pendante des anticorps. Cette revue d&eacute;crit les principaux types de r&eacute;cepteurs Fc et les voies de transduction de signal sont engag&eacute;s par eux dans les leucocytes diverses, en mettant l&#39;accent sur l&#39;activation de la phagocytose. On discute aussi des nouvelles d&eacute;couvertes sur le r&ocirc;le r&eacute;gulateur des r&eacute;cepteurs Fc sur la fonction immunitaire.

[Fonctions de r&eacute;cepteur fc r&eacute;gulation h&ocirc;te et immunitaire].

Recettori specifici per gli anticorpi, denominati recettori Fc sono proteine che si trovano sulla superficie dei leucociti. Recettori FC contribuiscono alle funzioni protettive del sistema immunitario, dall&#39;associazione di anticorpi che sono associati a cellule infette o patogeni. Attivit&agrave; del recettore FC stimola le cellule fagocitiche o citotossiche per distruggere i microbi, o infetta cellule di fagocitosi mediata da anticorpi o citotossicit&agrave; anticorpo-dipendente cellulo-mediata. Questa review descrive i principali tipi di recettori Fc e le vie di trasduzione del segnale che sono iniziate da li in vari leucociti, con enfasi sull&#39;attivazione della fagocitosi. Nuove scoperte sul ruolo normativo dei recettori Fc sulla funzione immunitaria inoltre sono discussi.

[Funzioni del recettore fc nel regolamento host e immunitario].

Receptores espec&iacute;ficos para el anticuerpo, llamado Fc receptores son prote&iacute;nas que se encuentran en la superficie de los leucocitos. Receptores FC contribuyen a las funciones protectoras del sistema inmunol&oacute;gico, por unir a los anticuerpos que se unen a las c&eacute;lulas infectadas o pat&oacute;genos invasores. La actividad del receptor FC estimula las c&eacute;lulas fagoc&iacute;ticas o citot&oacute;xicas para destruir microbios o infectado c&eacute;lulas por fagocitosis mediada por anticuerpos o la citotoxicidad mediada por c&eacute;lulas dependiente de anticuerpos. Esta revisi&oacute;n describe los principales tipos de receptores Fc y las v&iacute;as de transducci&oacute;n de se&ntilde;al que se inician por ellos en diferentes leucocitos, con &eacute;nfasis en la activaci&oacute;n de la fagocitosis. Tambi&eacute;n se discuten nuevos hallazgos en el papel regulador de receptores Fc en la funci&oacute;n inmune.

[Funciones de receptores fc en host and immune Reglamento].

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Amoebiasis, the disease caused by  is the third leading cause of human deaths among parasite infections.  was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010.  infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to  trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that  leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by  trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented -induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block -induced NETs formation.  induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that  activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

 Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Human neutrophils express two unique antibody receptors for IgG, the FcγRIIa and the FcγRIIIb. FcγRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcγRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcγRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcγRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcγRIIIb does not promote these responses. In contrast, FcγRIIIb has been reported to induce actin polymerization, activation of β1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcγRIIa. Another function where these receptors seem to act differently is the increase of cytoplasmic calcium concentration. It has been known for a long time that FcγRIIa induces production of inositol triphosphate (IP) to release calcium from intracellular stores, while FcγRIIIb does not use this phospholipid. Thus, the mechanism for FcγRIIIb-mediated calcium rise remains unknown. Transient Receptor Potential Melastatin 2 (TRPM2) is a calcium permeable channel expressed in many cell types including vascular smooth cells, endothelial cells and leukocytes. TRPM2 can be activated by protein kinase C (PKC) and by oxidative stress. Because we previously found that FcγRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated  FcγRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcγ receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcγRIIa induced a calcium rise coming initially from internal pools. In contrast, FcγRIIIb caused a calcium rise by inducing calcium entry from the extracellular medium. In addition, in the presence of 2-aminoethoxydiphenyl borate (2-APB) or of clotrimazole, two inhibitors of TRPM2, FcγRIIIb-induced calcium rise was blocked. fMLF- or FcγRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcγRIIIb aggregation activates TRPM2, to induce an increase in cytoplasmic calcium concentration through calcium internalization in human neutrophils.

The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry  Transient Receptor Potential Melastatin 2 in Human Neutrophils.

Receptors for FcγR on human neutrophils constitute an important mechanism for the recognition of opsonized microorganisms and for cell activation. Human neutrophils express 2 FcγR: FcγRIIa and FcγRIIIb. Previously, it has been reported that activation of each FcγR induces different neutrophil responses by triggering distinct signal transduction pathways, although what particular signal transduction pathway is triggered by each FcγR has not been completely elucidated. It has also been reported that PKC is important for FcγR signaling and that each FcγR may activate different PKC isoforms. Therefore, we explored whether FcγRIIa or FcγRIIIb activates different PKC isoforms in human neutrophils and whether activation of these PKC isoforms results in different neutrophil responses. Hence, either FcγRIIa or FcγRIIIb was selectively cross-linked by monoclonal antibodies in the presence or absence of pharmacological inhibitors for various PKC isoforms. Inhibition of PKCα or PKCδ blocked FcγRIIa-induced reactive oxygen species productions. In contrast, inhibition of PKCα and/or PKCβ blocked FcγRIIIb-induced reactive oxygen species production. Also, inhibition of all PKC isoforms did not affect the FcγRIIa-induced increase in intracellular calcium concentration ([Ca2+]i), while inhibition of PKCα blocked FcγRIIIb-induced increase in [Ca2+]i. Additionally, inhibition of PKCδ blocked FcγRIIa-induced ERK phosphorylation, while inhibition of PKCα prevented FcγRIIIb-induced ERK phosphorylation. These results suggest that both FcγRIIa and FcγRIIIb activate unique PKC isoforms and that activation of these PKC isoforms can selectively regulate different neutrophil functions. These findings also reinforce the idea that each FcγR in human neutrophils triggers distinct signal transduction pathways.

Article	Year
R-Ras promotes tumor growth of cervical epithelial cells. Cancer\| PubMed ID: 12548599	2003
R-Ras promotes metastasis of cervical cancer epithelial cells. Cancer immunology, immunotherapy : CII\| PubMed ID: 16862428	2007
FcgammaRIIA and FcgammaRIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19342628	2009
[Fc receptor functions in host and immune regulation]. Revista de investigaciÃ³n clÃnica; organo del Hospital de Enfermedades de la NutriciÃ³n\| PubMed ID: 19848309	2009
Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation. Journal of immunology research\| PubMed ID: 27034964	2016
Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation. Frontiers in immunology\| PubMed ID: 27486461	2016
Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation. Frontiers in cellular and infection microbiology\| PubMed ID: 30023352	2018
The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry Transient Receptor Potential Melastatin 2 in Human Neutrophils. Frontiers in immunology\| PubMed ID: 34054821	2021
Fc gamma receptors activate different protein kinase C isoforms in human neutrophils. Journal of leukocyte biology\| PubMed ID: 39946245	2025

Nancy Mora

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