Prospective association between pro-inflammatory state on admission and posttraumatic stress following acute coronary syndrome.

OBJECTIVE: The traumatic experience of acute coronary syndrome (ACS) may induce symptoms of posttraumatic stress disorder (PTSD). We examined whether the ACS-triggered acute inflammatory response predicts the development of PTSD symptoms. METHOD: Study participants were 70 patients (all Caucasian, 80% male, mean age 59 years) with myocardial infarction (MI) during the acute treatment phase. Interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, IL-4, IL-10, and transforming growth factor (TGF)-1ß were determined in plasma collected within 48 h of hospital admission. Participants self-assessed the severity of ACS-induced PTSD symptoms with the 17-item Posttraumatic Diagnostic Scale at 12 months. RESULTS: There was a significant positive association of the pro-inflammatory index (added standardized z-scores of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α) with total PTSD symptom severity (ΔR2 = 0.050, p = .029) and re-experiencing symptoms (ΔR2 = 0.088, p = .008), but not avoidance/numbing and hyperarousal symptoms. Analyses were adjusted for the anti-inflammatory index (added standardized z-scores of IL-4, IL-10, and TGF-ß1), trauma-focused counseling, sex, age, time since pain onset, troponin, body mass index, and distress during MI. Results were robust when the anti-inflammatory index was removed from the model. Additional analyses showed significant associations of both the net-inflammatory index (i.e., pro-inflammatory index minus anti-inflammatory index) and IL-1ß with total PTSD symptom severity, re-experiencing, and hyperarousal symptoms (ΔR2 between 0.042 and 0.090) and of IL-1ß with avoidance/numbing symptoms (ΔR2 = 0.050). CONCLUSIONS: The findings suggest an association between the pro-inflammatory state launched during ACS and the development of PTSD symptoms. Increased IL-1ß may play a particular role in the pathophysiology of ACS-induced PTSD symptoms.

CD25 as a unique marker on human basophils in stable-mildly symptomatic allergic asthma.

Background: Basophils in acute asthma exacerbation are activated as evidenced by their increased expression levels of activation markers such as CD203c and CD63. However, whether basophils of allergic asthmatics who are in stable phase and have no asthma exacerbations display a specific and distinctive phenotype from those of healthy individuals has yet to be well characterized. Objective: We aimed to identify the phenotype of basophils from allergic asthmatics in the stable phase and investigate whether such a phenotype is affected by ex vivo allergen stimulation. Methods: We determined by flow cytometry, the expression of surface proteins such as CD25, CD32, CD63, CD69, CD203c, and CD300a and intracellular anti-apoptotic proteins BCL-2, BCL-xL, and MCL-1. We investigated these markers in blood basophils obtained from well-characterized patients with stable-mildly symptomatic form of allergic asthma with no asthma exacerbation and from healthy individuals. Moreover, we determined ex vivo CD63, CD69, and CD25 on blood basophils from stable-mildly symptomatic allergic asthmatics upon allergen stimulation. Results: In contrast to all tested markers, CD25 was significantly increased on circulating basophils in the patient cohort with stable-mildly symptomatic allergic asthma than in healthy controls. The expression levels of CD25 on blood basophils showed a tendency to positively correlate with FeNO levels. Notably, CD25 expression was not affected by ex vivo allergen stimulation of blood basophils from stable-mildly symptomatic allergic asthma patients. Conclusion: Our data identifies CD25 as a unique marker on blood basophils of the stable phase of allergic asthma but not of asthma exacerbation as mimicked by ex vivo allergen stimulation.

Steroid hormone bioavailability is controlled by the lymphatic system.

The steroid hormone progesterone accounts for immune tolerance in pregnancy. Enhanced progesterone metabolism to 6α-OH-pregnanolone occurs in complicated pregnancies such as in preeclampsia with preterm delivery or intrauterine growth restriction, and in cancer. As lymphatic endothelial cells (LECs) promote tumor immunity, we hypothesized that human LECs modify progesterone bioavailability. Primary human LECs and mice lymph nodes were incubated with progesterone and progesterone metabolism was analyzed by thin layer chromatography and liquid chromatography-mass spectrometry. Expression of steroidogenic enzymes, down-stream signal and steroid hormone receptors was assessed by Real-time PCR. The placental cell line HTR-8/SV neo was used as reference. The impact of the progesterone metabolites of interest was investigated on the immune system by fluorescence-activated cell sorting analysis. LECs metabolize progesterone to 6α-OH-pregnanolone and reactivate progesterone from a precursor. LECs highly express 17ß-hydroxysteroid dehydrogenase 2 and are therefore antiandrogenic and antiestrogenic. LECs express several steroid hormone receptors and PIBF1. Progesterone and its metabolites reduced TNF-α and IFN-γ production in CD4+ and CD8+ T cells. LECs modify progesterone bioavailability and are a target of steroid hormones. Given the global area represented by LECs, they might have a critical immunomodulatory control in pregnancy and cancer.

Basophils Orchestrating Eosinophils' Chemotaxis and Function in Allergic Inflammation.

Eosinophils are well known to contribute significantly to Th2 immunity, such as allergic inflammations. Although basophils have often not been considered in the pathogenicity of allergic dermatitis and asthma, their role in Th2 immunity has become apparent in recent years. Eosinophils and basophils are present at sites of allergic inflammations. It is therefore reasonable to speculate that these two types of granulocytes interact in vivo. In various experimental allergy models, basophils and eosinophils appear to be closely linked by directly or indirectly influencing each other since they are responsive to similar cytokines and chemokines. Indeed, basophils are shown to be the gatekeepers that are capable of regulating eosinophil entry into inflammatory tissue sites through activation-induced interactions with endothelium. However, the direct evidence that eosinophils and basophils interact is still rarely described. Nevertheless, new findings on the regulation and function of eosinophils and basophils biology reported in the last 25 years have shed some light on their potential interaction. This review will focus on the current knowledge that basophils may regulate the biology of eosinophil in atopic dermatitis and allergic asthma.

IL-1ß promotes immunoregulatory responses in human blood basophils.

Human basophils are essential effector cells of chronic allergic inflammation. IL-1 family cytokines such as interleukin (IL)-33 and IL-1ß are elevated in serum and bronchoalveolar lavage fluid of allergic asthmatics. IL-33 is known to be a critical regulator of basophil's T2 immune responses. However, the effect of IL-1ß on the function of basophils has not been well investigated. Here, we elucidate whether IL-1ß regulates the function of human basophils and compared the effects of IL-1ß and IL-33 on basophils of healthy and allergic subjects. We found that IL-1ß activates the p38 MAPK signaling pathway and promotes IL-8 release in basophils of healthy donors, while FcεRI-mediated LCT4 and histamine secretion is not affected. Strikingly, in the presence of IL-3, IL-1ß shows more potency than IL-33, as evidenced by the enhanced p38 phosphorylation and NF-κB activation, as well as the release of both IL-13 and IL-8. We found that the enhanced basophil responsiveness is achieved through IL-3-induced IL-1RI surface expression. Importantly, basophils of allergic donors release significantly higher amounts of IL-8 compared to those from healthy donors upon IL-33 and IL-1ß stimulation. Consistently, we detected increased IL-1RI and decreased IL-3 receptor alpha-chain (CD123) and CCR3 expression on basophils of allergic subjects compared to healthy controls, suggesting an in vivo IL-3 priming in allergic donors. In summary, our results suggest enhanced sensitivity of basophils toward IL-33 and IL-1ß in allergic subjects compared to those from healthy controls.

Reduced Monocytic Human Leukocyte Antigen-DR Expression Indicates Immunosuppression in Critically Ill COVID-19 Patients.

BACKGROUND: The cellular immune system is of pivotal importance with regard to the response to severe infections. Monocytes/macrophages are considered key immune cells in infections and downregulation of the surface expression of monocytic human leukocyte antigen-DR (mHLA-DR) within the major histocompatibility complex class II reflects a state of immunosuppression, also referred to as injury-associated immunosuppression. As the role of immunosuppression in coronavirus disease 2019 (COVID-19) is currently unclear, we seek to explore the level of mHLA-DR expression in COVID-19 patients. METHODS: In a preliminary prospective monocentric observational study, 16 COVID-19-positive patients (75% male, median age: 68 [interquartile range 59-75]) requiring hospitalization were included. The median Acute Physiology and Chronic Health Evaluation-II (APACHE-II) score in 9 intensive care unit (ICU) patients with acute respiratory failure was 30 (interquartile range 25-32). Standardized quantitative assessment of HLA-DR on monocytes (cluster of differentiation 14+ cells) was performed using calibrated flow cytometry at baseline (ICU/hospital admission) and at days 3 and 5 after ICU admission. Baseline data were compared to hospitalized noncritically ill COVID-19 patients. RESULTS: While normal mHLA-DR expression was observed in all hospitalized noncritically ill patients (n = 7), 89% (8 of 9) critically ill patients with COVID-19-induced acute respiratory failure showed signs of downregulation of mHLA-DR at ICU admission. mHLA-DR expression at admission was significantly lower in critically ill patients (median, [quartiles]: 9280 antibodies/cell [6114, 16,567]) as compared to the noncritically ill patients (30,900 antibodies/cell [26,777, 52,251]), with a median difference of 21,508 antibodies/cell (95% confidence interval [CI], 14,118-42,971), P = .002. Reduced mHLA-DR expression was observed to persist until day 5 after ICU admission. CONCLUSIONS: When compared to noncritically ill hospitalized COVID-19 patients, ICU patients with severe COVID-19 disease showed reduced mHLA-DR expression on circulating CD14+ monocytes at ICU admission, indicating a dysfunctional immune response. This immunosuppressive (monocytic) phenotype remained unchanged over the ensuing days after ICU admission. Strategies aiming for immunomodulation in this population of critically ill patients should be guided by an immune-monitoring program in an effort to determine who might benefit best from a given immunological intervention.

Detection of Basophils and Other Granulocytes in Induced Sputum by Flow Cytometry.

Flow cytometry is one of the most widely used techniques for the detection of surface markers on various cells, particularly the cells of the immune system, at a single-cell resolution. Modern flow cytometers can identify rare cell population in highly heterogeneous samples. Here we present a protocol that allows a precise detection of basophils as well as eosinophils and neutrophils in induced sputum samples. The identification of sputum basophils and other granulocytes contributes to a better understanding of the cellular network that promotes and regulates inflammation of the lower respiratory tract.

Analysis of IL-6 serum levels and CAR T cell-specific digital PCR in the context of cytokine release syndrome.

INTRODUCTION: Chimeric antigen receptor T-cell (CAR-T) therapies are increasingly used to treat relapsed B-cell lymphomas and acute lymphoblastic leukemia. Considering the frequency of cytokine release syndrome and CAR-T-related encephalopathy syndrome (CRS/CRES) after CAR-T administration, strategies enabling timely prediction of impending CRS/CRES are a clinical need. METHODS: We evaluated the dynamics of serum interleukin (IL)-6 levels and CAR-T transgene copy numbers by digital droplet polymerase chain reaction in the peripheral blood of 11 consecutive patients with aggressive B-cell malignancies. RESULTS: Four of 11 patients developed CRS, and 3 patients had CRES (33%), 2 of them had previous CRS. IL-6 levels increased on the day of clinical manifestation of CRS. All CRS patients had increased IL-6 peak levels (median IL-6 peak 606 pg/mL in CRS patients vs. 22 pg/mL in non-CRS patients, p = 0.0061). Different patterns emerged from the dynamics of CAR-T/µg genomic DNA: "rapid increase and rapid decrease with complete disappearance," "rapid increase and slow decrease with higher persistence," "rapid increase and rapid decrease with lower persistence," and "slow increase and rapid decrease with almost disappearance." Patients with the pattern "rapid increase and slow decrease with higher persistence" of CAR-T/µg genomic DNA concentration seemed to be at higher risk of developing CRS/CRES. CONCLUSION: Thus, the dynamics of CAR-T transgene copy numbers merits further evaluation for a possible association with manifestation of CRS. Increased IL-6 serum levels at CRS manifestation may contribute to the interpretation of symptoms.

Impact of BH3-mimetics on Human and Mouse Blood Leukocytes: A Comparative Study.

BH3-mimetics are small molecule inhibitors that neutralize the function of anti-apoptotic BCL-2 family members. BH3-mimetics have recently gained a lot of popularity in oncology because of their success in cancer treatment. However, BH3-mimetics might have a broader clinical application. Here, we established an ex vivo flow cytometric assay allowing the comparison of the impact of BH3-mimetics (ABT-199, ABT-263, WEHI-539, and S63845) on leukocyte populations of both, healthy human subjects and C57BL/6 J wild type mice. BH3-mimetics were added to freshly drawn blood that was diluted 1/2 in cell medium, and BH3-mimetics-mediated impact on leukocyte count was assessed by flow cytometry. Our results demonstrate that responses towards 1µM of BH3-mimetics can be identical as well as considerably different in leukocytes of humans and mice. For instance, the inhibition of BCL-2 by ABT-199 caused cell death in all types of lymphocytes in mice but was exclusively specific for B cells in humans. Moreover, inhibition of BCL-XL by WEHI-539 affected solely mouse leukocytes while targeting MCL-1 by S63845 resulted in efficient induction of cell death in human neutrophils but not in their mouse counterparts. Our ex vivo assay enables initial identification of analogies and differences between human and mouse leukocytes in response towards BH3-mimetics.

Evaluation of polyvinylpyrrolidone and block copolymer micelle encapsulation of serine chlorin e6 and chlorin e4 on their reactivity towards albumin and transferrin and their cell uptake.

Encapsulation of porphyrinic photosensitizers (PSs) into polymeric carriers plays an important role in enhancing their efficiency as drugs in photodynamic therapy (PDT). Porphyrin aggregation and low solubility as well as the preservation of the advantageous photophysical properties pose a challenge on the design of efficient PS-carrier systems. Block copolymer micelles (BCMs) and polyvinylpyrrolidone (PVP) are promising drug delivery vehicles for physical entrapment of PSs. BCMs exhibit enhanced dynamics as compared to the less flexible PVP network. In the current work the question is addressed how these different dynamics affect PS encapsulation, release from the carrier, reaction with serum proteins, and cellular uptake. The porphyrinic compounds serine-amide of chlorin e6 (SerCE) and chlorin e4 (CE4) were used as model PSs with different lipophilicity and aggregation properties. 1H NMR and fluorescence spectroscopy were applied to study their interactions with PVP and BCMs consisting of Kolliphor P188 (KP). Both chlorins were well encapsulated by the carriers and had improved photophysical properties. Compared to SerCE, the more lipophilic CE4 exhibited stronger hydrophobic interactions with the BCM core, stabilizing the system and preventing exchange with the surrounding medium as was shown by NMR NOESY and DOSY experiments. PVP and BCMs protected the encapsulated chlorins against interaction with human transferrin (Tf). However, SerCE and CE4 were released from BCMs in favor of binding to human serum albumin (HSA) while PVP prevented interaction with HSA. Fluorescence spectroscopic studies revealed that HSA binds to the surface of PVP forming a protein corona. PVP and BCMs reduced cellular uptake of the chlorins. However, encapsulation into BCMs resulted in more efficient cell internalization for CE4 than for SerCE. HSA significantly lowered both, free and carrier-mediated cell uptake for CE4 and SerCE. In conclusion, PVP appears as the more universal delivery system covering a broad range of host molecules with respect to polarity, whereas BCMs require a higher drug-carrier compatibility. Poorly soluble hydrophobic PSs benefit stronger from BCM-type carriers due to enhanced bioavailability through disaggregation and solubilization allowing for more efficient cell uptake. In addition, increased PS-carrier hydrophobic interactions have a stabilizing effect. For more hydrophilic PSs, the main advantage of polymeric carriers like PVP or poloxamer micelles lies in their protection during the transport through the bloodstream. HSA binding plays an important role for drug release and cell uptake in carrier-mediated delivery to the target tissue.

Nutrient pattern analysis in critically ill patients using Omics technology (NAChO) - Study protocol for a prospective observational study.

INTRODUCTION: Intensive care unit-acquired weakness (ICU-AW) is often observed in critically ill patients with prolonged intensive care unit (ICU) stay. We hypothesized that evolving metabolic abnormalities during prolonged ICU stay are reflected by changing nutrient patterns in blood, urine and skeletal muscle, and that these patterns differ in patients with/without ICU-AW and between patients with/without sepsis. METHODS: In a prospective single-center observational trial, we aim to recruit 100 critically ill patients (ICU length of stay ≥ 5 days) with severe sepsis/septic shock ("sepsis group", n = 50) or severe head trauma/intracerebral hemorrhage ("CNS group", n = 50). Patients will be sub-grouped for presence or absence of ICU-AW as determined by the Medical Research Council sum score. Blood and urine samples will be collected and subjected to comprehensive nutrient analysis at different time points by targeted quantitative mass spectrometric methods. In addition, changes in muscular tissue (biopsy, when available), muscular architecture (ultrasound), electrophysiology, body composition analyses (bioimpedance, cerebral magnetic resonance imaging), along with clinical status will be assessed. Patients will be followed-up for 180 and 360 days including assessment of quality of life. DISCUSSION: Key objective of this trial is to assess changes in nutrient pattern in blood and urine over time in critically ill patients with/without ICU-AW by using quantitative nutrient analysis techniques. Peer-reviewed published NAChO data will allow for a better understanding of metabolic changes in critically ill patients on standard liquid enteral nutrition and will likely open up new avenues for future therapeutic and nutritional interventions.

Allogeneic hematopoietic cell transplantation in patients with GATA2 deficiency-a case report and comprehensive review of the literature.

Recently, an immunodeficiency syndrome caused by guanine-adenine-thymine-adenine 2 (GATA2) deficiency has been described. The syndrome is characterized by (i) typical onset in early adulthood, (ii) profound peripheral blood cytopenias of monocytes, B lymphocytes, and NK cells, (iii) distinct susceptibility to disseminated non-tuberculous mycobacterial (NTM) and other opportunistic infections (particularly human papillomavirus), and (iv) a high risk of developing hematologic malignancies (myelodysplastic syndromes (MDS); acute myeloid leukemias (AML)). Considerable clinical heterogeneity exists among patients with GATA2 deficiency, but once infectious symptoms occur or MDS/AML arises, survival declines significantly. Allogeneic hematopoietic cell transplantation (HCT) currently provides the only curative treatment option for both MDS/AML and dysfunctional immunity with life-threatening opportunistic infections. Strategies regarding timing of allogeneic HCT, antimicrobial prophylaxis and treatment, intensity of the preparative regimen, and optimal donor and graft source have not been clearly defined due to the rarity of the disease. Here, we provide a comprehensive analysis of the available literature and published case reports on the use of allogeneic HCT in patients with GATA2 deficiency. In addition, a case of a young woman with GATA2 deficiency, who developed an immune reconstitution inflammatory syndrome in her mycobacterial skin lesions post allogeneic HCT is presented and illustrates distinct problems encountered in this disease context.

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells.

Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.

Are Inflammatory Cytokines Associated with Pain during Acute Myocardial Infarction?

OBJECTIVE: Pain and inflammation during acute myocardial infarction (AMI) have been associated with the development of posttraumatic stress disorder and may also impact negatively on somatic outcome. We investigated the relationship between pain during AMI and levels of circulating proinflammatory (tumor necrosis factor [TNF]-α, interleukin [IL]-6) and anti-inflammatory (IL-33 and tissue growth factor [TGF]-ß1) cytokines. METHODS: Data were collected as part of the Myocardial Infarction - Stress Prevention Intervention (MI-SPRINT) study. We included 140 patients (mean age 59.6 years, 82.1% male) with high acute psychological distress within 48 h after MI. Fasting blood samples were drawn thereafter to measure cytokine levels. Sociodemographic factors, psychological and medical data, as well as cardiometabolic markers were assessed with questionnaires and patient interviews. RESULTS: Linear regression models showed a significant positive correlation of pain with TGF-ß1 (b = 770.91, p = 0.031) and a significant inverse correlation of pain with IL-33 (b = -0.11, p = 0.015) after controlling for age, gender, body mass index, lifetime depression, acute stress disorder symptoms, and the prognostic Global Registry of Acute Coronary Events (GRACE) score. Pain was not associated with IL-6 but with the GRACE score (b = 0.01, p = 0.003). Pain showed no significant association with TNF-α. CONCLUSION: Pain during MI was associated with anti- but not proinflammatory cytokines. As IL-33 has been shown to be cardioprotective, lower IL-33 levels with more intense pain may suggest a pathway through which increased pain during MI may have an impact on the medical prognosis.

Assessment of immune organ dysfunction in critical illness: utility of innate immune response markers.

In critically ill patients, organ dysfunctions are routinely assessed, monitored, and treated. Mounting data show that substantial critical illness-induced changes in the immune system can be observed in most ICU patients and that not only "hyper-inflammation" but also persistence of an anti-inflammatory phenotype (as in sepsis-associated immunosuppression) is associated with increased morbidity and mortality. Despite common perception, changes in functional immunity cannot be adequately assessed by routine inflammatory biomarkers such as C-reactive protein, procalcitonin, or numerical analysis of leukocyte (sub)-counts. Cytokines appear also not suited due to their short half-life and pleiotropy, their unexclusive origin from immune cells, and their potential to undergo antagonization by circulating inactivating molecules. Thus, beyond leukocyte quantification and use of routine biomarkers, direct assessment of immune cell function seems required to characterize the immune systems' status. This may include determination of, e.g., ex vivo cellular cytokine release, phagocytosis activity, and/or antigen-presenting capacity. In this regard, standardized flow-cytometric assessment of the major histocompatibility-II complex human leukocyte antigen (-D related) (HLA-DR) has gained particular interest. Monocytic HLA-DR (mHLA-DR) controls the interplay between innate and adaptive immunity and may serve as a "global" biomarker of injury-associated immunosuppression, and its decreased expression is associated with adverse clinical outcomes (e.g., secondary infection risk, mortality). Importantly, recent data demonstrate that injury-associated immunosuppression can be reversed-opening up new therapeutic avenues in affected patients. Here we discuss the potential scientific and clinical value of assessment of functional immunity with a focus on monocytes/macrophages and review the current state of knowledge and potential perspectives for affected critically ill patients.

Late IL-3-induced phenotypic and functional alterations in human basophils require continuous IL-3 receptor signaling.

Cytokines of the GM-CSF family signal via the same receptor subunit (ßc) and, thus, have overlapping effects on cells that express all cytokine-specific α-chains (IL-3Rα, IL-5Rα, GM-CSFRα), such as human basophils, whose rapid effector functions are similarly enhanced by IL-3, IL-5, and GM-CSF. However, previous work has shown that IL-3, but not IL-5 and GM-CSF, supports and induces allergy-associated functions of human basophils at later time points. This includes induction of Th2 cytokine and chemokine secretion, high-affinity IgE receptor-independent leukotriene C4 (LTC4) formation, expression of enzymes (e.g., RALDH2, granzyme B), and kinases (e.g., Pim1). Here, we address the question of why IL-3, but not IL-5 or GM-CSF, is capable of inducing these late responses in human basophils, and we investigate the mechanism that underlies the unique regulatory capacity of IL-3. We find that IL-3, IL-5, and GM-CSF rapidly activate the same canonical signaling cascades in a qualitatively identical manner with comparable strength, but we identify signaling duration as major discriminating factor. IL-5 and GM-CSF rapidly down-regulate surface levels of their receptors within minutes, concomitant with a rapid decay in signaling molecule activation and time-dependent loss of ability of these cytokines to prime basophils for functional responses. By contrast, IL-3 hardly down-regulates the α-chain of its receptor without depleting the common ß-chain, which enables extraordinarily sustained signaling events, predominantly the activation of Stat5. Of interest, acute IL-3 signaling is not sufficient to induce persistent phenotypical and functional changes in human basophils. Induction of these functional late responses depends on continuous IL-3 receptor activation and signaling.

Macrophage-independent T cell infiltration to the site of injury-induced brain inflammation.

We have addressed the role of macrophages in glial response and T cell entry to the CNS after axonal injury, by using intravenous injection of clodronate-loaded mannosylated liposomes, in C57BL6 mice. As expected, clodronate-liposome treatment resulted in depletion of peripheral macrophages which was confirmed by F4/80- and MOMA-1(-) stainings in spleen. Sequential clodronate-liposome treatment 4, 2 and 0 days before axotomy resulted in significant reduction of infiltrating CD45(high) CD11b+ macrophages in the hippocampus at 1, 2 and 3 days post-lesion, measured by flow cytometry. There was a slight delay in the expansion of CD45(dim) CD11+ microglia in clodronate-liposome treated mice, but macrophage depletion had no effect on the percentage of infiltrating T cells in the lesion-reactive hippocampus. Lesion-induced TNFalpha mRNA expression was not affected by macrophage depletion, suggesting that activated glial cells are the primary source of this cytokine in the axonal injury-reactive brain. This identifies a potentially important distinction from inflammatory autoimmune infiltration in EAE, where macrophages are a prominent source of TNFalpha and their depletion prevents parenchymal T cell infiltration and disease.