Creating and Testing a CD207+ Langerhans Cell Histiocytosis-like Cell Line

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) activation. Investigations into LCH have historically been challenged by the small percentage of pathologic CD207+ DCs. No cell lines with morphology or function representing LCH lesion CD207+ DCs currently exist. We utilized four strategies to generate cell line(s) mimicking differentiated LCH pathogenic cells. First, CD207+ cells were isolated from the lesion of an LCH patient and cultured in a cytokine cocktail. The cells maintained CD1a and CD207 expression for two weeks, after which there were significant changes in cell morphology and progression to cell death. When a similar approach was implemented to isolate and culture skin CD207+ cells, the cells were viable for only three days, supporting the potential role for somatic activating MAPK mutations in LCH lesion DCs to prolong viability. CD207+ cells were then isolated from HLA-DR+ (lineage negative) cells from the lymphoid stroma of healthy tonsils (tCD207+). The tCD207+ cells were transduced with a lentivirus encoding human telomerase reverse transcriptase (hTERT). These cells, also lacking MAPK activating mutations, died two weeks post-transduction. A fourth approach has been more successful in which tCD207+ cells were cultured in a cytokine cocktail which provided MAPK pathway stimulation. The cells retained CD207 expression and survived in culture for over two weeks. The cells were then immortalized using a lentivirus encoding HOXA9. Immortalized cells maintained CD207 expression. Allele specific MAPK pathway mutations (BRAF and MAP2K1) are being generated by class II CRISPR/Cpf1 genome editing as Cpf1 has been shown to have robust activity to induce specific disruption of only mutant, but not wild-type, BRAF allele. The phenotypic and genomic characteristics of the immortalized cells expressing the different MAPK pathway mutations will be assessed by RHG-banding cytogenetic analysis, fluorescence in situ hybridization, gene expression analysis, and immuno‐cytochemistry and results will be compared to cells isolated from LCH lesions to confirm whether the established cell line may be a viable in vitro mimic of the LCH lesion DC. Disclosures No relevant conflicts of interest to declare.

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RecurrentNRASmutations in pulmonary Langerhans cell histiocytosis

European Respiratory Journal ◽

10.1183/13993003.01677-2015 ◽

2016 ◽

Vol 47 (6) ◽

pp. 1785-1796 ◽

Cited By ~ 49

Author(s):

Samia Mourah ◽

Alexandre How-Kit ◽

Véronique Meignin ◽

Dominique Gossot ◽

Gwenaël Lorillon ◽

...

Keyword(s):

Protein Kinase ◽

Lung Tissue ◽

Langerhans Cell Histiocytosis ◽

Kinase Inhibitor ◽

Mapk Pathway ◽

Univariate Analysis ◽

Langerhans Cell ◽

Mitogen Activated Protein Kinase ◽

Normal Lung ◽

Pulmonary Langerhans Cell Histiocytosis

The mitogen-activated protein kinase (MAPK) pathway is constantly activated in Langerhans cell histiocytosis (LCH). Mutations of the downstream kinasesBRAFandMAP2K1mediate this activation in a subset of LCH lesions. In this study, we attempted to identify other mutations which may explain the MAPK activation in nonmutatedBRAFandMAP2K1LCH lesions.We analysed 26 pulmonary and 37 nonpulmonary LCH lesions for the presence ofBRAF,MAP2K1,NRASandKRASmutations. Grossly normal lung tissue from 10 smoker patients was used as control. Patient spontaneous outcomes were concurrently assessed.BRAFV600Emutations were observed in 50% and 38% of the pulmonary and nonpulmonary LCH lesions, respectively. 40% of pulmonary LCH lesions harbouredNRASQ61K/Rmutations, whereas noNRASmutations were identified in nonpulmonary LCH biopsies or in lung tissue control. In seven out of 11NRASQ61K/R-mutated pulmonary LCH lesions,BRAFV600Emutations were also present. Separately genotyping each CD1a-positive area from the same pulmonary LCH lesion demonstrated that these concurrentBRAFandNRASmutations were carried by different cell clones.NRASQ61K/Rmutations activated both the MAPK and AKT (protein kinase B) pathways. In the univariate analysis, the presence of concurrentBRAFV600EandNRASQ61K/Rmutations was significantly associated with patient outcome.These findings highlight the importance ofNRASgenotyping of pulmonary LCH lesions because the use of BRAF inhibitors in this context may lead to paradoxical disease progression. These patients might benefit from MAPK kinase inhibitor-based treatments.

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Transcriptomic Landscape of Circulating Mononuclear Phagocytes in Langerhans Cell Histiocytosis at Single-cell Level

Blood ◽

10.1182/blood.2020009064 ◽

2021 ◽

Author(s):

Hui Shi ◽

Han He ◽

Lei Cui ◽

Egle Kvedaraite ◽

Zhilei Bian ◽

...

Keyword(s):

Single Cell ◽

Langerhans Cell Histiocytosis ◽

Mapk Pathway ◽

Braf Inhibitor ◽

Clinical Diagnostics ◽

Langerhans Cell ◽

Mononuclear Phagocytes ◽

Mitogen Activated Protein Kinase ◽

Erk Signaling

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm caused by aberrant activation of the mitogen-activated protein kinase (MAPK) pathway. Circulating myeloid cells from patients often carry disease-associated mutations and can be differentiated into langerinhigh LCH-like cells in vitro, but their detailed immune-phenotypic and molecular profiles are lacking and could shed key insights into disease biology. Here we recruited 217 pediatric LCH patients and took blood and tissue samples for BRAFV600E analysis. Immune-phenotyping of the circulating Lin-HLA-DR+ immune population in 49 of these patients revealed that decreased frequency of pDC was significantly linked to disease severity. By single-cell RNA sequencing of samples from 14 patients, we identified key changes in expression of RAS-MAPK-ERK signaling-related genes and transcription factors in distinct members of the mononuclear phagocyte system in the presence of BRAFV600E. Moreover, treatment of patients with the BRAF inhibitor Dabrafenib resulted in MAPK cascade inhibition, inflammation prevention, and regulation of cellular metabolism within mononuclear phagocytes. Finally, we also observed elevated expression of RAS-MAPK-ERK signaling-related genes in a CD207+CD1a+ cell subcluster in skin. Taken together, our data extends the molecular understanding of LCH biology at single-cell resolution, which might contribute to improvement of clinical diagnostics and therapeutics, and aid in the development of personalized medicine approaches.

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RAF/MEK/extracellular signal–related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions

Journal of Experimental Medicine ◽

10.1084/jem.20161881 ◽

2017 ◽

Vol 215 (1) ◽

pp. 319-336 ◽

Cited By ~ 15

Author(s):

Brandon Hogstad ◽

Marie-Luise Berres ◽

Rikhia Chakraborty ◽

Jun Tang ◽

Camille Bigenwald ◽

...

Keyword(s):

Dendritic Cells ◽

Langerhans Cell Histiocytosis ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Langerhans Cell ◽

Mitogen Activated Protein Kinase ◽

Phagocytic Cells ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Myeloid Neoplasia

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal–related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)–mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.

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How I manage pulmonary Langerhans cell histiocytosis

European Respiratory Review ◽

10.1183/16000617.0070-2017 ◽

2017 ◽

Vol 26 (145) ◽

pp. 170070 ◽

Cited By ~ 15

Author(s):

Gwenaël Lorillon ◽

Abdellatif Tazi

Keyword(s):

Langerhans Cell Histiocytosis ◽

Mapk Pathway ◽

Therapeutic Option ◽

Significant Proportion ◽

Airflow Obstruction ◽

Forced Expiratory Volume ◽

Langerhans Cell ◽

Mitogen Activated Protein Kinase ◽

Pulmonary Langerhans Cell Histiocytosis ◽

Right Heart Catheterisation

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare sporadic cystic lung disease of unknown aetiology that is characterised by the infiltration and destruction of the wall of distal bronchioles by CD1a+Langerhans-like cells. In adults, PLCH is frequently isolated and affects young smokers of both sexes. Recent multicentre studies have led to the more standardised management of patients in clinical practice. Smoking cessation is essential and is occasionally the only suitable intervention. Serial lung function testing is important because a significant proportion of patients may experience an early decline in forced expiratory volume in 1 s and develop airflow obstruction. Cladribine was reported to dramatically improve progressive PLCH in some patients. Its efficacy and tolerance are currently being evaluated. Patients who complain of unexplained dyspnoea with decreased diffusing capacity of the lung for carbon monoxide should be screened for pulmonary hypertension by Doppler echocardiography, which must be confirmed by right heart catheterisation. Lung transplantation is a therapeutic option for patients with advanced PLCH.The identification of theBRAFV600Emutation in approximately half of Langerhans cell histiocytosis lesions, including PLCH, and other mutations of the mitogen-activated protein kinase (MAPK) pathway in a subset of lesions has led to targeted treatments (BRAF and MEK (MAPK kinase) inhibitors). These treatments need to be rigorously evaluated because of their potentially severe side-effects.

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Biomodulatory Metronomic Therapy Shows Remarkable Activity in Chemorefractory Multi-System Langerhans Cell Histiocytosis

Blood ◽

10.1182/blood.v128.22.4254.4254 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4254-4254 ◽

Cited By ~ 1

Author(s):

Daniel Heudobler ◽

Klaus Rehe ◽

Juergen Foell ◽

Selim Corbacioglu ◽

Gerhard Hildebrandt ◽

...

Keyword(s):

Complete Remission ◽

Langerhans Cell Histiocytosis ◽

Low Dose ◽

Conflicts Of Interest ◽

Mapk Pathway ◽

Prospective Trial ◽

Langerhans Cell ◽

University Hospital ◽

Response To Treatment ◽

Metronomic Therapy

Abstract Background: Langerhans cell histiocytosis (LCH) is a disorder characterized by lesions that include CD207+ dendritic cells along with an inflammatory infiltrate, ranging from a single lesion to potentially fatal multi-system disease. Empirically derived chemotherapy with vinblastine and prednisone still represents the standard of care, curing fewer than 50% of patients. As a result of the discovery of activating mutations in the MAPK-pathway, BRAF inhibitors seem to be a novel therapeutic option. In LCH patients who were refractory to (or not eligible for) conventional chemotherapy we administered a multi-targeted therapeutic approach targeting the deregulation of homeostatic pathways in LCH, e.g. notch, thereby simultaneously modulating tumor-associated inflammation, angiogenesis and immune response. The present retrospective analysis reports a single-center experience with a biomodulatory therapy in a cohort of patients with refractory LCH. Methods: At the University Hospital Regensburg seven patients with multi-system LCH (2 female/5 male, including two pediatric patients), ranging from 11 months to 77 years old, were selected for compassionate use treatment with a biomodulatory therapy schedule (Br J Haematol. 2005;128:730-2). Six patients have been refractory to up to three different standard regimens. One patient was not eligible for conventional therapy due to severe comorbidities. The biomodulatory metronomic therapy consisted of low dose trofosfamide, pioglitazone, a PPAR alpha/gamma agonist, etoricoxib, a COX-2 inhibitor and low-dose dexamethasone. Each drug was administered daily until disease progression or in case of complete remission for additional 6 months. Treatment response was reported using the criteria established in the Histiocyte Society Evaluation and Treatment Guidelines. Results: All of the patients in our cohort showed response to treatment as determined clinically, by CT/MRI scans and re-biopsies. Two of them developed a complete remission, three regression of disease and two stable disease. To date, none of the patients developed progressive disease (representing a progression-free-survival of 10 to 55 months until now). In one patient a dose reduction had to be made due to leukopenia. No other significant adverse events have been observed. Conclusion: Biomodulatory metronomic therapy demonstrated high activity against multi-system LCH with minimal toxicity even in refractory patients following multiple systemic chemotherapies. Long-lasting disease control was achieved in all of the treated patients. Confirmation of efficacy should be evaluated in a prospective trial. Disclosures No relevant conflicts of interest to declare.

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Three decades of progress from surgery to medical therapy for isolated neuroaxis BRAF V600E–positive Langerhans cell histiocytosis management: illustrative case

Journal of Neurosurgery: Case Lessons ◽

10.3171/case2118 ◽

2021 ◽

Vol 1 (19) ◽

Author(s):

Nallammai Muthiah ◽

Kamil W. Nowicki ◽

Jennifer L. Picarsic ◽

Michael P. D’Angelo ◽

Daniel F. Marker ◽

...

Keyword(s):

Langerhans Cell Histiocytosis ◽

Mapk Pathway ◽

Clinical Manifestations ◽

Tumor Burden ◽

Langerhans Cell ◽

Mitogen Activated Protein Kinase ◽

Braf V600e ◽

Illustrative Case ◽

Surgical Interventions ◽

Mitogen Activated Protein

BACKGROUND “Langerhans cell histiocytosis” (LCH) is a term that encompasses single-system or multisystem disorders traditionally characterized by a proliferation of clonal CD1a+/CD207+ myeloid-derived histiocytes. In most cases of LCH, mitogen-activated protein kinase (MAPK) pathway somatic mutations lead to near universal upregulation of phosphorylated extracellular signal-regulated kinase expression. The clinical manifestations of LCH are numerous, but bone involvement is common. Intracranial lesions, especially as isolated manifestations, are rare. OBSERVATIONS The authors presented the case of a long-term survivor of exclusive intracranial LCH that manifested with isolated craniofacial bone and intraparenchymal central nervous system recurrences, which were managed with 3 decades of multimodal therapy. The patient was initially diagnosed with LCH at age 2 years, and the authors documented the manifestations of disease and treatment for 36 years. Most of the patient’s treatment course occurred before the discovery of BRAF V600E. Treatments initially consisted of chemotherapy, radiosurgery, and open resections for granulomatous LCH lesions. Into young adulthood, the patient had a minimal disease burden but still required additional radiosurgical procedures and open resections. LESSONS Surgical treatments alleviated the patient’s immediate symptoms and allowed for tumor burden control. However, surgical interventions did not cure the underlying, aggressive disease. In the current era, access to systemic MAPK inhibitor therapy for histiocytic lesions may offer improved outcomes.

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Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

Acta Endocrinologica ◽

10.1530/eje.0.1510233 ◽

2004 ◽

pp. 233-240 ◽

Cited By ~ 99

Author(s):

AM Nanzer ◽

S Khalaf ◽

AM Mozid ◽

RC Fowkes ◽

MV Patel ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Kinase ◽

Cell Line ◽

Kinase Inhibitor ◽

Thymidine Incorporation ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Gh3 Cells ◽

Rat Pituitary ◽

Mitogen Activated Protein

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.

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Establishment of cell line and in vivo mouse model of canine Langerhans cell histiocytosis

Veterinary and Comparative Oncology ◽

10.1111/vco.12476 ◽

2019 ◽

Vol 17 (3) ◽

pp. 345-353 ◽

Cited By ~ 1

Author(s):

Nguyen V. Son ◽

Kazuyuki Uchida ◽

Atigan Thongtharb ◽

James K. Chambers ◽

Takuya E. Kishimoto ◽

...

Keyword(s):

Mouse Model ◽

Cell Line ◽

Langerhans Cell Histiocytosis ◽

Langerhans Cell

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Pulmonary Langerhans Cell Histiocytosis

Seminars in Respiratory and Critical Care Medicine ◽

10.1055/s-0039-1700996 ◽

2020 ◽

Vol 41 (02) ◽

pp. 269-279

Author(s):

Brian Shaw ◽

Michael Borchers ◽

Dani Zander ◽

Nishant Gupta

Keyword(s):

Langerhans Cell Histiocytosis ◽

Treatment Options ◽

Skin Lesions ◽

Langerhans Cell ◽

Mitogen Activated Protein Kinase ◽

Myeloid Neoplasm ◽

Cystic Lung ◽

Pulmonary Langerhans Cell Histiocytosis ◽

Mitogen Activated Protein ◽

The Central Nervous System

AbstractPulmonary Langerhans cell histiocytosis (PLCH) is a diffuse cystic lung disease that is strongly associated with exposure to cigarette smoke. Recently, activating pathogenic mutations in the mitogen-activated protein kinase pathway have been described in the dendritic cells in patients with PLCH and have firmly established PLCH to be an inflammatory myeloid neoplasm. Disease course and prognosis in PLCH are highly variable among individual patients, ranging from spontaneous resolution to development of pulmonary hypertension and progression to terminal respiratory failure. A subset of patients with PLCH may have extrapulmonary involvement, typically involving the skeletal system in the form of lytic lesions, skin lesions, or the central nervous system most commonly manifesting in the form of diabetes insipidus. Smoking cessation is the cornerstone of treatment in patients with PLCH and can lead to disease regression or stabilization in a substantial proportion of patients. Further insight into the underlying molecular pathogenesis of PLCH has paved the way for the future development of disease-specific biomarkers and targeted treatment options directed against the central disease-driving mutations.

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Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP’s Growth-Inhibitory Effects

Blood ◽

10.1182/blood.v93.2.537 ◽

1999 ◽

Vol 93 (2) ◽

pp. 537-553 ◽

Cited By ~ 26

Author(s):

Angel Wai-mun Lee

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Cell Line ◽

Mapk Pathway ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Erk Activation ◽

Mitogen Activated Protein ◽

Erk Activity ◽

Myeloid Progenitors

Abstract Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90rsk. cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1–mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.

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