High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

Potentiation of cytokine-induced proliferation of human Natural Killer cells by intravenous immunoglobulin G.

Intravenous IgG (IVIG) therapy can be used for immunomodulation. IL-2 is an immunoregulatory cytokine. We evaluated IVIG modulation of human blood lymphocyte response to IL-2 and other cytokines. Neither IVIG nor low concentrations of IL-2 (3-30U/ml) induced lymphocyte proliferation, but in combination they synergistically enhanced proliferation of NK cells. The CD56(bright) cells expanded more than CD56(dim) NK cells, with 90% of NK cells dividing up to 8 generations by day 6, while <8% of T cells divided. IVIG also potentiated NK cell proliferation with IL-12, IL-15 and IL-18. The IVIG+cytokine-expanded NK cells were less cytotoxic for K562 cells, than NK cells with cytokine alone. IVIG also enhanced interferon-γ production with IL-2, IL-12 and IL-18. In conclusion, IVIG selectively potentiates NK cell proliferation and interferon-γ secretion with IL-2, IL-12, IL-15 and IL-18 in vitro. These findings warrant evaluation in vivo in relation to NK cells and the immunoregulatory actions of IVIG.

Intravenous IgG (IVIG) and subcutaneous IgG (SCIG) preparations have comparable inhibitory effect on T cell activation, which is not dependent on IgG sialylation, monocytes or B cells.

IVIG modulates T cell activation in vitro and inflammatory-autoimmune conditions in vivo. Sialylation of IgG, Fc receptor interactions, modulation of monocyte/macrophage/B cell functions have been implicated in IVIG effects. Subcutaneous IgG (SCIG) therapy is increasingly used for IgG replacement but whether these preparations share the effects of IVIG on T cell modulation is not documented. We compared the potency of SCIG-Hizentra™ (20% IgG preparation) with IVIG-Privigen® (10% IgG) for T cell inhibition, and assessed the involvement of IgG sialylation, monocytes and B cells in this process. Human PBMCs or sorted cells were cultured 3-7 days, and T cells were stimulated with immobilized anti-CD3 mAb or Candida antigen. Thymidine incorporation into DNA was quantitated and cytokines assayed by ELISA/Luminex® assay. IVIG and SCIG both dose-dependently (1-20mg/ml) inhibited (up to >80%) T cell proliferation to anti-CD3 mAb. Response to Candida albicans was comparably inhibited by IVIG and SCIG by 50-80% at 10mg/ml with inhibition even at 3mg/ml (P<0.05). These effects were not affected by depletion of sialic acid containing IgG using neuraminidase treatment or lectin affinity chromatography. With anti-CD3 or Candida stimulation, IL-1ß, IL-2, IL-5, IL-6, IL-13, GMCSF, TNF-α, interferon-γ (with anti-CD3) and IL-17 (with Candida) levels were suppressed by IVIG or SCIG, with no effect on IL-4, IL-10, IL-12, IL-15 or TGFß. Monocytes or B cells were not required for IgG-induced suppression of proliferation, in fact depletion of monocytes potentiated the IgG-induced inhibition. Reconstitution with monocytes restored the original inhibitory effect. These data show that IVIG (Privigen®) and SCIG (Hizentra™) have comparable inhibitory effects on T cell activation, which do not require sialylation of IgG. Inhibition is independent of monocytes or B cells. There is a potent suppression of multiple effector cytokines. Like IVIG, SCIG therapy is expected to show immunomodulatory activity.

Fatal pneumococcal meningitis in a 7-year-old girl with interleukin-1 receptor activated kinase deficiency (IRAK-4) despite prophylactic antibiotic and IgG responses to Streptococcus pneumoniae vaccines.

UNLABELLED: IRAK-4 deficiency causes IL-1R and TLR signaling failure, resulting in minimal clinical features despite invasive bacterial infection. We report the course of a 7-year-old IRAK-4-deficient girl presenting in the first year with multiple occult Staphylococcus aureus lymphadenitis. She was managed with antibiotic prophylaxis (sulfa/trimethoprim/PenV, then - due to neutropenia - Cefprozil), pneumococcal vaccination (PCV-7, Pneumovax23, PCV-13) and vigilance. Pneumococcal-specific IgG levels were monitored. No bacterial infections occurred on prophylaxis for 6 years after initial presentation. IgG response to pneumococcal polysaccharide was satisfactory but short-lived, requiring frequent boosting. At age 7, patient developed a morning headache and vomited once. Cefprozil was administered and re-dosed. Over 12 h, she was fatigued without other symptoms. Low fever accompanied another emesis. A few hours later she was confused, and purpuric rash appeared. Emergency physicians diagnosed sepsis/meningitis and started vancomycin-ceftriaxone. Respiratory failure and cerebellar herniation occurred <24 h after first symptoms. Blood and CSF grew Streptococcus pneumoniae type 6C resistant to second-generation cephalosporins. The patient's latest PCV-13 vaccination was 6 weeks before death, which included serotype 6A. Immunoglobulins were normal except IgG4 was increased (3.4 g/L). IgG response to vaccine antigens was satisfactory. IgG to 6A is reported to cross-react with 6C, but this was not the case here. CONCLUSION: Despite antibiotic prophylaxis and repeated vaccination, even older IRAK-4-deficient patients are at high risk of rapidly fatal infection due to emergence of antibiotic resistance. These patients need early assessment at any age, bacterial culturing, alternative empiric antibiotic therapy and close observation when even vaguely unwell. Based on increasingly recognized immunological and/or clinical impairments in B cell function, and possibly other defects, long-term IgG prophylaxis in addition to antibiotics is recommended.

IgM+IgD+CD27+ B cells are markedly reduced in IRAK-4-, MyD88-, and TIRAP- but not UNC-93B-deficient patients.

We studied the distribution of peripheral B-cell subsets in patients deficient for key factors of the TLR-signaling pathways (MyD88, TIRAP/MAL, IL-1 receptor-associated kinase 4 [IRAK-4], TLR3, UNC-93B, TRIF). All TLRs, except TLR3, which signals through the TRIF adaptor, require MyD88 and IRAK-4 to mediate their function. TLR4 and the TLR2 heterodimers (with TLR1, TLR6, and possibly TLR10) require in addition the adaptor TIRAP, whereas UNC-93B is needed for the proper localization of intracellular TLR3, TLR7, TLR8, and TLR9. We found that IgM(+)IgD(+)CD27(+) but not switched B cells were strongly reduced in MyD88-, IRAK-4-, and TIRAP-deficient patients. This defect did not appear to be compensated with age. However, somatic hypermutation of Ig genes and heavy-chain CDR3 size distribution of IgM(+)IgD(+)CD27(+) B cells were not affected in these patients. In contrast, the numbers of IgM(+)IgD(+)CD27(+) B cells were normal in the absence of TLR3, TRIF, and UNC-93B, suggesting that UNC-93B-dependent TLRs, and notably TLR9, are dispensable for the presence of this subset in peripheral blood. Interestingly, TLR10 was found to be expressed at greater levels in IgM(+)IgD(+)CD27(+) compared with switched B cells in healthy patients. Hence, we propose a role for TIRAP-dependent TLRs, possibly TLR10 in particular, in the development and/or maintenance of IgM(+)IgD(+)CD27(+) B cells in humans.

Differing requirements for CCR4, E-selectin, and α4ß1 for the migration of memory CD4 and activated T cells to dermal inflammation.

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)ß(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)ß(1) was more important. Anti-α(4)ß(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)ß(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)ß(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)ß(1), and CCR4(-) cells preferentially use α(4)ß(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

Bilateral adrenal EBV-associated smooth muscle tumors in a child with a natural killer cell deficiency.

EBV-associated smooth muscle tumors are found in immunocompromised patients, most commonly HIV/AIDS. We present a 12-year-old girl with the first documented case of EBV-related smooth muscle tumors in the presence of a rare classic NK cell deficiency. This sheds light on the role of NK cells in controlling EBV-related smooth muscle tumors.

Intravenous immunoglobulin G (IVIG) inhibits IL-1- and TNF-α-dependent, but not chemotactic-factor-stimulated, neutrophil transendothelial migration.

High-dose intravenous immunoglobulin (IVIG) has anti-inflammatory effects via incompletely understood mechanisms. By investigating whether IVIG might modulate neutrophil (PMN) recruitment, we observed that IVIG dose-dependently inhibited (by 30-50%) PMN transendothelial migration (TEM) across human umbilical vein endothelial cells (EC) stimulated with IL-1α, IL-1ß, TNF-α or IL-1ß+TNF-α. Inhibition required the presence of IVIG with the responding PMNs, was attributable to the F(ab)(2) portion and was unrelated to putative contaminants in IVIG. IVIG did not inhibit IL-1ß- or TNF-α-induced increase of PMN adhesion to EC, nor did it affect C5a- or IL-8-induced PMN TEM across unstimulated EC. Effects of IVIG and F(ab)(2) fragments were not associated with PMN activation, assessed by CD62L shedding, CD11b upregulation or PMN shape. Thus, IVIG selectively inhibits PMN TEM across inflammatory-cytokine-stimulated - but not unstimulated - EC, perhaps contributing to therapeutic benefit in chronic inflammation with minimal impact on chemotactic-factor-induced PMN recruitment during acute infection.

Coexpression of chemokine receptors CCR5, CXCR3, and CCR4 and ligands for P- and E-selectin on T lymphocytes of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate P- and E-selectin ligand coexpression with chemokine receptors (CKRs) on T cells in the synovial fluid (SF) and blood of children with juvenile idiopathic arthritis (JIA). METHODS: Sixteen patients with polyarticular or persistent oligoarticular JIA (ages 5.3-15.1 years) were studied. SF and venous blood were collected, and immunostaining for the expression of CCR4, CCR5, CXCR3, and P- or E-selectin ligands was performed. RESULTS: Compared to blood, SF was greatly enriched for CD4+ T cells bearing CCR5, CCR4, CXCR3, and both P- and E-selectin ligand. Twenty-five percent of the CD4+ T cells in SF expressed both CCR5 and CCR4, some also coexpressing CXCR3. Such cells were rare in blood. Half of the few CCR5+ T cells in blood coexpressed P- or E-selectin ligand, a phenotype that was enriched up to 50-fold in SF. A minority of CCR4+ and CXCR3+ cells in blood (∼25%) coexpressed selectin ligand; these were enriched 4-8-fold in SF. Most CCR4-expressing CD4+ T cells expressed both E-selectin ligand and cutaneous lymphocyte antigen. CONCLUSION: CCR4-, CCR5-, CXCR3-, and selectin ligand-expressing CD4+ T cells preferentially accumulate in the joints of children with JIA. The marked enrichment of CCR5+ T cells coexpressing P-selectin and/or E-selectin ligand in CD4+ SF T cells suggests that the few such cells in blood selectively migrate to inflamed joints via endothelial P- and E-selectin- and CCR5-activating chemokines. The predominance of CCR4-expressing CD4+ T cells coexpressing E-selectin ligand suggests that such cells migrate not only to areas of cutaneous inflammation, as previously reported, but also to the joints in JIA. Combined targeting of CCR5- and E-selectin-dependent mechanisms may be a relevant treatment strategy.

Myeloid dysplasia and bone marrow hypocellularity in adenosine deaminase-deficient severe combined immune deficiency.

Genetic deficiency of adenosine deaminase (ADA) can cause profound lymphopenia and result in the clinical presentation of severe combined immune deficiency (SCID). However, because of the ubiquitous expression of ADA, ADA-deficient patients often present also with nonimmunologic clinical problems, affecting the skeletal, central nervous, endocrine, and gastrointestinal systems. We now report that myeloid dysplasia features and bone marrow hypocellularity are often found in patients with ADA-SCID. As a clinical correlate to this finding, we have observed vulnerability to antibiotic-induced myelotoxicity and prolonged neutropenia after nonmyeloablative chemotherapy. We have also noted that, in the absence of enzyme replacement therapy, absolute neutrophil counts of patients with ADA deficiency vary inversely with the accumulation of deoxynucleotides. These data have significant implications for the application of standard and investigational therapies to patients with ADA-SCID and support further studies to investigate the possibility that ADA deficiency is associated with a stem cell defect. These trials were registered at www.clinicaltrials.gov as #NCT00018018 and #NCT00006319.

Dengue virus infection of mast cells triggers endothelial cell activation.

Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection.

Clinical features and outcome of patients with IRAK-4 and MyD88 deficiency.

Autosomal recessive interleukin-1 receptor-associated kinase (IRAK)-4 and myeloid differentiation factor (MyD)88 deficiencies impair Toll-like receptor (TLR)- and interleukin-1 receptor-mediated immunity. We documented the clinical features and outcome of 48 patients with IRAK-4 deficiency and 12 patients with MyD88 deficiency, from 37 kindreds in 15 countries.The clinical features of IRAK-4 and MyD88 deficiency were indistinguishable. There were no severe viral, parasitic, and fungal diseases, and the range of bacterial infections was narrow. Noninvasive bacterial infections occurred in 52 patients, with a high incidence of infections of the upper respiratory tract and the skin, mostly caused by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The leading threat was invasive pneumococcal disease, documented in 41 patients (68%) and causing 72 documented invasive infections (52.2%). P. aeruginosa and Staph. aureus documented invasive infections also occurred (16.7% and 16%, respectively, in 13 and 13 patients, respectively). Systemic signs of inflammation were usually weak or delayed. The first invasive infection occurred before the age of 2 years in 53 (88.3%) and in the neonatal period in 19 (32.7%) patients. Multiple or recurrent invasive infections were observed in most survivors (n = 36/50, 72%).Clinical outcome was poor, with 24 deaths, in 10 cases during the first invasive episode and in 16 cases of invasive pneumococcal disease. However, no death and invasive infectious disease were reported in patients after the age of 8 years and 14 years, respectively. Antibiotic prophylaxis (n = 34), antipneumococcal vaccination (n = 31), and/or IgG infusion (n = 19), when instituted, had a beneficial impact on patients until the teenage years, with no seemingly detectable impact thereafter.IRAK-4 and MyD88 deficiencies predispose patients to recurrent life-threatening bacterial diseases, such as invasive pneumococcal disease in particular, in infancy and early childhood, with weak signs of inflammation. Patients and families should be informed of the risk of developing life-threatening infections; empiric antibacterial treatment and immediate medical consultation are strongly recommended in cases of suspected infection or moderate fever. Prophylactic measures in childhood are beneficial, until spontaneous improvement occurs in adolescence.

Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Trans- but not cis-resveratrol impairs angiotensin-II-mediated vascular inflammation through inhibition of NF-κB activation and peroxisome proliferator-activated receptor-gamma upregulation.

Angiotensin II (Ang-II) displays inflammatory activity and is implicated in several cardiovascular disorders. This study evaluates the effect of cis- and trans (t)-resveratrol (RESV) in two in vivo models of vascular inflammation and identifies the cardioprotective mechanisms that underlie them. In vivo, Ang-II-induced arteriolar leukocyte adhesion was inhibited by 71% by t-RESV (2.1 mg/kg, i.v.), but was not affected by cis-RESV. Because estrogens influence the rennin-angiotensin system, chronic treatment with t-RESV (15 mg/kg/day, orally) inhibited ovariectomy-induced arteriolar leukocyte adhesion by 81%, partly through a reduction of cell adhesion molecule (CAM) expression and circulating levels of cytokine-induced neutrophil chemoattractant, MCP-1, and MIP-1alpha. In an in vitro flow chamber system, t-RESV (1-10 microM) undermined the adhesion of human leukocytes under physiological flow to Ang-II-activated human endothelial cells. These effects were accompanied by reductions in monocyte and endothelial CAM expression, chemokine release, phosphorylation of p38 MAPK, and phosphorylation of the p65 subunit of NF-kappaB. Interestingly, t-RESV increased the expression of peroxisome proliferator-activated receptor-gamma in human endothelial and mononuclear cells. These results demonstrate for the first time that the in vivo anti-inflammatory activity of RESV is produced by its t-RESV, which possibly interferes with signaling pathways that cause the upregulation of CAMs and chemokine release. Upregulation of proliferator-activated receptor-gamma also appears to be involved in the cardioprotective effects of t-RESV. In this way, chronic administration of t-RESV may reduce the systemic inflammatory response associated with the activation of the rennin-angiotensin system, thereby decreasing the risk of further cardiovascular disease.

Impaired T-cell receptor activation in IL-1 receptor-associated kinase-4-deficient patients.

BACKGROUND: IL-1 receptor-associated kinase 4 (IRAK-4) is an effector of the Toll-like receptor and IL-1 receptor pathways that plays a critical role in innate immune responses. The role of IRAK-4 in adaptive immune functions in human subjects is incompletely understood. OBJECTIVE: We sought to evaluate T-cell function in IRAK-4 deficient patients. METHODS: We compared upregulation of CD25 and CD69 on T cells and production of IL-2, IL-6, and IFN-gamma after stimulation of PBMCs from 4 IRAK-4-deficient patients and healthy control subjects with anti-CD3 and anti-CD28. RESULTS: Upregulation of CD25 and CD69 on T cells and production of IL-6 and IFN-gamma, but not IL-2, was significantly reduced in IRAK-4-deficient patients. CONCLUSIONS: IRAK-4-deficient patients have defects in T-cell activation.

Intravenous immunoglobulin G selectively inhibits IL-1α-induced neutrophil-endothelial cell adhesion.

OBJECTIVES: Intravenous immunoglobulin (IVIG) G at high doses has therapeutic benefits in a variety of autoimmune and inflammatory disorders. The mechanism by which IVIG modulates inflammation is incompletely understood. We tested the hypothesis that IVIG modulates inflammation by inhibiting interactions between neutrophils and vascular endothelium, required for leukocyte recruitment to inflamed tissues. METHODS: The adhesion of human blood neutrophils to resting or cytokine-activated human umbilical vein endothelial cells (HUVECs) was measured, and the effect of IVIG or normal donor sera added at various stages was determined. RESULTS: IVIG completely inhibited neutrophil adhesion to endothelium stimulated with interleukin-1 (IL-1α), when it was present during the endothelial stimulation phase. IVIG had no effect on adhesion when IL-1ß or TNF-α was the activating cytokine. The plasma of some (one of five) healthy donors also selectively blocked the IL-1α activation of the endothelium for supporting adhesion, and this was due to the presence of neutralizing IgG at high levels in the blood of the donor. CONCLUSIONS: Thus, IgG antibodies to IL-1α are present in IVIG at a biologically significant level, which can prevent endothelial activation. However, IVIG does not directly affect activation of endothelium or neutrophil adhesion mechanisms. The anti-inflammatory properties of IVIG may in part be related to blocking IL-1α-dependent leukocyte recruitment. Potentially, such antibodies may also have immunoregulatory effects by binding and neutralizing membrane-bound IL-1α during cell-cell interactions.

Menopause and ovariectomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan.

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-beta-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1alpha and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1alpha in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-beta-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function.

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

Intravenous immunoglobulin G-mediated inhibition of T-cell proliferation reflects an endogenous mechanism by which IgG modulates T-cell activation.

Commercial intravenous immunoglobulin G (IVIG) at high doses has therapeutic benefit in autoimmune and inflammatory diseases. It has been shown to inhibit T-cell function but the mechanisms are unclear. Inhibition could result from IVIG processing, donor pooling or intrinsic downregulatory activity of IgG. To address these points, we compared the effects on T-cell activation of IVIG, Fab(2) fragment and IgG isolated from single-donor plasma. We also investigated the role of accessory cells in the IVIG effects using highly purified T cells stimulated through CD3 and CD28 engagement. T-cell proliferation was evaluated by Oregon Green 488 dye dilution and (3)H-thymidine incorporation. IVIG, Fab(2) fragment of IVIG and autologous, single-donor IgG significantly inhibited T-cell proliferation (35-50%), even in the absence of accessory cells. Depletion of IgG from plasma used for culture significantly increased (by 50%) the T-cell proliferation. The addition of physiological concentrations of single-donor, autologous IgG or IVIG to IgG-depleted plasma reduced T-cell proliferation to levels observed in normal plasma. Therefore, donor pooling in IVIG and accessory cells are not required for inhibition of T-cell proliferation by IVIG and the Fab(2) region is sufficient to mediate this inhibition. Suppression of T-cell activation by IVIG likely reflects a physiologic, endogenous mechanism of IgG-mediated regulation of T-cell activation.

Cellular players and role of selectin ligands in leukocyte recruitment in a T-cell-initiated delayed-type hypersensitivity reaction.

Delayed-type hypersensitivity (DTH) reactions are characterized by a strong cellular infiltrate, including neutrophils, macrophages, and T lymphocytes. In all these cell types, both E- and P-selectin-dependent adhesion pathways play a significant role in recruitment into the inflamed skin. Accordingly, inhibition of selectin-mediated interactions (eg, by antibodies) results in impairment of acute DTH reactions. However, whether inhibition of a specific cell type is responsible for the anti-inflammatory effect or whether all leukocytes are affected remains unclear. To address this question, we used fucosyltransferase-VII knockout mice that lack functional selectin ligands as either donors or recipients in a DTH model elicited by Th1 cell and antigen transfer. We found that selectin-mediated adhesion is required by Th1 effector cells to enter the DTH reaction site and, additionally, to elicit the DTH reaction. On the other hand, elimination of selectin binding in the recipient's neutrophils and macrophages by use of fucosyltransferase-deficient mice receiving wild-type Th1 effector cells resulted in a strongly reduced infiltration of neutrophils and macrophages but unimpaired footpad swelling. These findings demonstrate a major role for both E- and P-selectin in the recruitment of different leukocyte cell types. However, only the presence of selectin ligands on T cells was critical for the inflammatory reaction. These findings reveal T cells as the predominant targets for selectin blockade that aim to suppress skin inflammation.