Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2.

The immunologic consequences of prolonged infusions of rIL-2 in doses that produce physiologic serum concentrations of this cytokine were investigated. rIL-2 in doses of 0.5-6.0 x 10(6) U/m2 per d (3.3-40 micrograms/m2 per d) was administered by continuous intravenous infusion for 90 consecutive days to patients with advanced cancer. IL-2 concentrations (25 +/- 25 and 77 +/- 64 pM, respectively) that selectively saturate high-affinity IL-2 receptors (IL-2R) were achieved in the serum of patients receiving rIL-2 infusions of 10 micrograms/m2 per d and 30 micrograms/m2 per d. A gradual, progressive expansion of natural killer (NK) cells was seen in the peripheral blood of these patients with no evidence of a plateau effect during the 3 mo of therapy. A preferential expansion of CD56bright NK cells was consistently evident. NK cytotoxicity against tumor targets was only slightly enhanced at these dose levels. However, brief incubation of these expanded NK cells with IL-2 in vitro induced potent lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Infusions of rIL-2 at 40 micrograms/m2 per d produced serum IL-2 levels (345 +/- 381 pM) sufficient to engage intermediate affinity IL-2R p75, which is constitutively expressed by human NK cells. This did not result in greater NK cell expansion compared to the lower dose levels, but did produce in vivo activation of NK cytotoxicity, as evidenced by lysis of NK-resistant targets. There was no consistent change in the numbers of CD56- CD3+ T cells, CD56+ CD3+ MHC-unrestricted T cells, or B cells during infusions of rIL-2 at any of the dosages used. This study demonstrates that prolonged infusions of rIL-2 in doses that saturate only high affinity IL-2R can selectively expand human NK cells for an extended period of time with only minimal toxicity. Further activation of NK cytolytic activity can also be achieved in vivo, but it requires concentrations of IL-2 that bind intermediate affinity IL-2R p75. Clinical trials are underway attempting to exploit the differing effects of various concentrations of IL-2 on human NK cells in vivo.

Mental stimulation increases circulating CD4-positive T lymphocytes: a preliminary study.

To stimulate the dorsolateral frontal cortex, 12 healthy, adult, human females played contract bridge for 1.5 h between initial and final blood sample collections. Flow cytometric analyses of samples, performed in triplicate, showed a significant increase in CD4-positive T lymphocytes. The dorsolateral frontal cortical thickness is significantly and bilaterally reduced in immune-incompetent female, nude mice. Thymic transplants reverse the deficient cortical thickness and CD4-positive cell numbers.

Splenic artery aneurysm-pancreatic duct fistula.

Massive hemorrhage as a sequela of chronic pancreatitis may originate from many sources. This report documents a rare case of communication between the pancreatic ductal system and a pseudoaneurysm of the splenic artery. The multidisciplinary management of this unusual case required the combined expertise of the endoscopist, radiologist and surgeon.

Fatal liver disease associated with alpha 1-antitrypsin deficiency PiM1/PiMduarte.

A 53-yr-old man with a rare form of partial alpha 1-antitrypsin deficiency, PiM1/PiMduarte, died of endstage cirrhosis. Typical cytoplasmic alpha 1-antitrypsin globules were present in the hepatocyte cytoplasm. Initial protease inhibitor phenotyping on the patient was reported as normal PiM1 in more than one laboratory. This case emphasizes the diagnostic importance of alpha 1-antitrypsin and illustrates the point that protease inhibitor phenotyping without family genotyping may be misleading in heterozygous patients with liver disease.

Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells.

Human natural killer (NK) cells can be subdivided into two populations based on the density of cell surface CD56 antigen. The great majority (approximately 90%) of NK cells express CD56 at low levels (the CD56dim phenotype), whereas a small NK cell subset (approximately 10%) exhibits approximately fivefold greater density of surface CD56. Exposure to exogenous interleukin 2 (IL 2) induces tenfold greater proliferation of CD56bright cells compared to CD56dim lymphocytes, even though both subsets constitutively express similar levels of intermediate affinity IL 2 receptor (IL 2R) p75 chains. Incubation with IL 2 alone or irradiated target cells alone could induce expression of the IL 2R p55 chain by both CD56bright and CD56dim NK cells; a combination of both stimuli was most effective. IL 2R p55 induction was evident after co-culture of NK cells with both NK-sensitive and NK-resistant cell lines or with antibody-coated target cells. Activation of NK cells with IL 2 plus target cells resulted in enhanced proliferation compared to activation with IL 2 alone; target cells alone did not induce significant proliferation. Although both NK cell subsets appeared to express high-affinity IL 2R p75/p55 heterodimers after stimulation with target cells and IL 2, proliferation of CD56dim cells remained minimal after such activation; activated CD56dim cells consistently demonstrated less proliferation to IL 2 than did resting CD56bright cells. In contrast, CD56bright NK cells exhibited even greater proliferation after stimulation with target cells. Almost all CD56dim NK cells expressed CD16 (Fc gamma R III) as well as the NK zeta chain, whereas less than 50% of CD56bright cells express either CD16 or zeta. CD56bright and CD56dim lymphocytes, thus, appear to represent distinct subpopulations of NK cells with different functional activities. Unlike CD56bright cells, CD56dim NK cells do not proliferate optimally to IL 2, even after the latter have been stimulated to express both IL 2R p55 and IL 2R p75. Efficient proliferation of CD56dim NK cells may, thus, require additional or alternative signals.

Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.

Follicular dendritic cells inhibit human B-lymphocyte proliferation.

In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1. When irradiated FDCs were cultured with mitogen stimulated B cells, B cell 3H-TdR uptake was inhibited by up to 80%. This inhibitory effect was not seen when paraformaldehyde fixed FDCs were added to B-cell cultures, suggesting that the FDCs needed to be metabolically active. Moreover, supernatants from cultured FDCs were similarly able to inhibit B-cell proliferation. These results demonstrate that FDCs may downregulate the clonal expansion of B cells that occurs within lymphoid follicles as part of the normal physiologic immune response. Potentially, the loss of the inhibitory role of FDCs in vivo may be of importance in certain infectious and neoplastic processes in which germinal centers are affected.

Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) can produce prolonged clinical remission in some patients with hematologic malignancies. Unfortunately, disease relapse may occur despite BMT. Studies in animal models and clinical experience have provided evidence that immunologic factors play an important role in preventing relapse post-BMT. To stimulate immunologic activity in patients post-BMT, we administered prolonged uninterrupted continuous infusions of low-dose recombinant interleukin-2 (rIL-2). Thirteen marrow recipients (seven autologous BMT, six CD6 T-depleted allogeneic BMT) received rIL-2 at a dose of 2 x 10(5) U/m2/d for a scheduled period of 90 days. rIL-2 was administered through a Hickman catheter with a portable pump beginning a median of 85 days after BMT. Toxicity was minimal and all treatment could be undertaken in the outpatient setting. No patient developed any signs of graft-versus-host disease, hypotension, or pulmonary capillary leak syndrome. Treatment did not affect the absolute neutrophil count or hemoglobin level, but eosinophils increased substantially in most patients. Platelet counts decreased by 20% in 10 of 13 individuals within 2 weeks, but stabilized thereafter. Despite the low dose of rIL-2 administered, significant immunologic changes were noted. Specifically, all 13 patients experienced a marked increase (fivefold to 40-fold) in natural killer (NK) cell number. Phenotypic characterization showed that the majority of NK cells were CD56bright+ CD16+ CD3-. In contrast, a minor increase in T-cell number was noted in only 4 of 13 patients. Low-dose rIL-2 treatment resulted in augmentation of in vitro cytotoxicity against K562 and COLO tumor targets. This cytotoxic activity could be dramatically enhanced by incubation with additional rIL-2 in vitro. The immunologic effects of rIL-2 treatment were similar in both autologous and allogeneic marrow recipients. Our data suggest that prolonged infusion of rIL-2 at low doses is safe and can selectively increase NK cell number and activity after BMT. Further studies to assess the impact these changes may have on disease relapse post-BMT will be undertaken.

Overexpression of the human 2-oxoglutarate carrier lowers mitochondrial membrane potential in HEK-293 cells: contrast with the unique cold-induced mitochondrial carrier CGI-69.

Using differential mRNA expression analysis, a previously uncharacterized gene was found to be up-regulated 2-fold in brown adipose tissue (BAT) of mice exposed to cold (4 degrees C) for 48 h. Contig and homology analysis revealed that the gene represents the murine orthologue to a sequence from a public database encoding a putative human protein (CGI-69). The presence of mitochondrial carrier domains in the human protein, its transmembrane topology and cold-induction of the mouse CGI-69 gene in BAT prompted an analysis of the idea that CGI-69 may represent a new uncoupling protein (UCP) functional homologue. However, transfection of human CGI-69 isoforms in HEK-293 cells yielded no change in mitochondrial membrane potential (Deltapsi(m)), despite localization of FLAG-tagged CGI-69 to mitochondria of MCF7 cells. Surprisingly, overexpression of the human 2-oxoglutarate carrier (OGC) protein (originally designed as a negative control) sparked a significant drop in Deltapsi(m), possibly signalling a previously unappreciated uncoupling activity for the OGC.

Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation.

Mitochondrial uncoupling proteins have been implicated in the maintenance of metabolic rate and adaptational thermoregulation. We recently reported the identification of a brain-specific mitochondrial uncoupling protein homologue, UCP4. Here we characterized another newly described member of the uncoupling protein family, termed UCP5 (also called BMCP1). UCP5 transcripts are present in multiple human and mouse tissues, with an especially high abundance in the brain and testis. Expression of UCP5 in mammalian cells reduces the mitochondrial membrane potential. Multiple isoforms of UCP5 were identified and exhibited tissue-specific distribution and different potency in reduction of membrane potential. Furthermore, the mRNA abundance of both UCP4 and UCP5 is modulated by nutritional status or temperature in a tissue-specific manner in mice. Brain UCP4 and UCP5 mRNA transcripts rose by 1.5- and 1.7-fold, respectively, and liver UCP5 expression increased by 1.8-fold in response to acute cold exposure. A high-fat diet increased UCP5 mRNA in liver by 1.6-fold selectively in the obesity-resistant A/J but not in the obesity-prone C57BL/6J mouse strain. Liver UCP5 expression decreased significantly with a 24 h fast and was restored to the normal level after refeeding. In contrast, brain transcripts for both genes were not significantly altered by fasting or high-fat diet. These findings are consistent with the notion that UCP4 and UCP5 may be involved in tissue-specific thermoregulation and metabolic changes associated with nutritional status.

Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation.

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.

Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8.

Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitro required DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation.

Forced expression of murine IL-17E induces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice.

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-gamma in several tissues and elevated serum TNF-alpha were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.