The Effect of a Monoclonal Antibody Coupled to Ricin A Chain-Derived Peptides on Endothelial Cells in Vitro: Insights into Toxin-Mediated Vascular Damage

Immunotoxins (ITs) containing plant or bacterial toxins have a dose-limiting toxicity of vascular leak syndrome (VLS) in humans. The active A chain of ricin toxin (RTA), other toxins, ribosome-inactivating proteins, and the VLS-inducing cytokine IL-2 contain the conserved sequence motif (x)D(y) where X = L, I, G, or V and Y = V, L, or S. RTA-derived LDV-containing peptides attached to a monoclonal antibody, RFB4, induce endothelial cell (EC) damage in vitro and vascular leak in two animal models in vivo. We have now investigated the mechanism(s) by which this occurs and have found that (1) the exposed D75 in the LDV sequence in RTA and the C-terminal flanking threonine play critical roles in the ability of RFB4-conjugated RTA peptide to bind to and damage ECs and (2) the LDV sequence in RTA induces early manifestations of apoptosis in HUVECs by activating caspase-3. These data suggest that RTA-mediated inhibition of protein synthesis (due to its active site) and apoptosis (due to LDV) may be mediated by different portions of the RTA molecule. These results suggest that ITs prepared with RTA mutants containing alterations in LDVT may kill tumor cells in vivo in the absence of EC-mediated VLS.     

Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T1/2 of 263 h, the mcRFB4s had T1/2s ranging from 39 to 106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.     

Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T_1/2 of 263 h, the mcRFB4s had T_1/2s ranging from 39–106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T_1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.Key words: chimeric, anti-CD22, monoclonal antibody, Fc mutations, ricin A chain, immunotoxins     

Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD₅₀ values of wt and mut1 in mice were investigated with the result that the LD₅₀ of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use. Hao Wang, Shuichuan Song and Geng Kou contributed equally to this paper.     

Evidence for the involvement of CD44 in endothelial cell injury and induction of vascular leak syndrome by IL-2.

At sites of chronic inflammation seen during infections, autoimmunity, graft-vs-host response, and cytokine therapy, endothelial cell injury is known to occur, the exact mechanism of which is unknown. In the current study we used IL-2-induced vascular leak syndrome (VLS) as a model to investigate whether cytotoxic lymphocytes use CD44 in mediating endothelial cell injury. Administration of IL-2 to wild-type mice triggered significant VLS in the lungs and liver. In contrast, in CD44 knockout (KO) mice, IL-2-induced VLS was markedly reduced in the lungs and liver. IL-2-treated wild-type and CD44 KO mice had similar levels of perivascular infiltration with lymphocytes in the lungs and liver. This suggested that the decrease in VLS seen in CD44 KO mice was not due to the inability of lymphocytes to migrate to these organs. Ultrastructural studies demonstrated extensive endothelial cell damage in the lungs and liver of IL-2-treated wild-type, but not CD44 KO, mice. Moreover, CD44-KO mice exhibited a marked decrease in IL-2-induced lymphokine-activated killer cell activity. The induction of VLS was dependent on the expression of CD44 on immune cells rather than endothelial cells because adoptive transfer of CD44+, but not CD44- spleen cells along with IL-2 into CD44 KO mice triggered VLS. The IL-2-induced VLS was blocked by administration of F(ab')2 of Abs against CD44. The current study demonstrates that CD44 plays a key role in endothelial cell injury. Blocking CD44 in vivo may offer a novel therapeutic approach to prevent endothelial cell injury by cytotoxic lymphocytes in a variety of clinical disease models.     

Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice

Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three-amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.     

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome

IL-2-induced vascular leak syndrome (VLS) is an important mechanism explaining the toxic effects of this cytokine and limiting its therapeutic use. We previously characterized a mouse model of IL-2-induced pulmonary VLS used to demonstrate that NK lymphocytes are involved in early/acute phase VLS (after one IL-2 injection). We also showed that NK cells and polymorphonuclear neutrophils (PMN) are involved in the late/chronic phase of the syndrome (after four daily IL-2 injections). In this study we use our mouse model to evaluate the role played by the IL-2 receptor (IL-2R) in VLS induction. Mouse and human IL-2R are different since the mouse IL-2Rbeta chain does not recognize IL-2. Here, we compare the acute and late VLS responses in human IL-2Rbeta transgenic and C57BL/6 wild type mice. Parameters linked to early phase VLS (bronchoconstriction and PMN mobilization) are enhanced in human IL-2Rbeta transgenic mice. By contrast, parameters used to measure late events (protein leakage and edema) are similar in human IL-2Rbeta transgenic mice and C57BL/6 wild type animals. However, after four IL-2 injections, the cellular content of the bronchoalveolar lavage fluids was different between the two types of animals. This study also characterizes a humanized animal model that could be further used to study human IL-2 activity and side effects in vivo.     

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome

IL-2-induced vascular leak syndrome (VLS) is an important mechanism explaining the toxic effects of this cytokine and limiting its therapeutic use. We previously characterized a mouse model of IL-2-induced pulmonary VLS used to demonstrate that NK lymphocytes are involved in early/acute phase VLS (after one IL-2 injection). We also showed that NK cells and polymorphonuclear neutrophils (PMN) are involved in the late/chronic phase of the syndrome (after four daily IL-2 injections). In this study we use our mouse model to evaluate the role played by the IL-2 receptor (IL-2R) in VLS induction. Mouse and human IL-2R are different since the mouse IL-2Rbeta chain does not recognize IL-2. Here, we compare the acute and late VLS responses in human IL-2Rbeta transgenic and C57BL/6 wild type mice. Parameters linked to early phase VLS (bronchoconstriction and PMN mobilization) are enhanced in human IL-2Rbeta transgenic mice. By contrast, parameters used to measure late events (protein leakage and edema) are similar in human IL-2Rbeta transgenic mice and C57BL/6 wild type animals. However, after four IL-2 injections, the cellular content of the bronchoalveolar lavage fluids was different between the two types of animals. This study also characterizes a humanized animal model that could be further used to study human IL-2 activity and side effects in vivo.     

Blockade of hyaluronan inhibits IL-2-induced vascular leak syndrome and maintains effectiveness of IL-2 treatment for metastatic melanoma

Vascular leak syndrome (VLS) is a life-threatening toxicity induced during IL-2 treatment of cancer patients. The mechanism of IL-2-induced VLS is still poorly understood. At present, there is no specific therapy for VLS. Previous studies from our laboratory demonstrated that hyaluronan (HA), a large glycosaminoglycan, abundant in the extracellular matrix and on the cell surface, caused a marked increase of IL-2-induced VLS in the lungs and liver of C57BL/6 mice. Conversely, blockade or knockout of its major receptor, CD44, resulted in a marked decrease of VLS, thereby suggesting a role for HA in VLS. In this study, we report a novel means to prevent IL-2-induced VLS by blocking endogenous HA with HA-specific binding peptide, Pep-1, a newly isolated peptide which specifically binds to soluble, cell-associated, and immobilized forms of HA. Our results demonstrated that blocking HA with Pep-1 dramatically inhibited IL-2-induced VLS in both normal mice as well as in mice bearing melanoma. Moreover, Pep-1 treatment maintained the effectiveness of IL-2 and prevented the metastasis of melanoma. IL-2-induced emigration of lymphocytes across the endothelium and cytotoxicity against tumor by lymphokine-activated killer cells were not affected by Pep-1. Instead, use of Pep-1 maintained endothelial integrity and reduced their apoptosis during IL-2-induced VLS. These data suggested that HA plays a critical role in regulating endothelial cell damage and induction of IL-2-mediated VLS. Also, blockade of HA using Pep-1 could constitute a novel therapeutic modality to prevent IL-2-mediated toxicity, thereby facilitating the effectiveness of high-dose IL-2 in the treatment of metastatic melanomas.     

Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin

It has been shown that the alpha 4 beta 1 integrin is the lymphocyte receptor for the carboxy terminal cell-binding domain of fibronectin which comprises adhesion sites in Hep 2 and a high affinity site, CS-1, in the type III connecting segment or V (for variable) region. In the present studies, using a series of peptides derived from CS-1, we identify the tripeptide leu-asp-val (LDV), as the minimal peptide capable of supporting stable lymphocyte or melanoma cell adhesion. However, only cells which expressed an active form of the alpha 4 beta 1 complex were capable of attaching to and spreading on LDV peptide. On a molar basis, LDV minimal peptides were either not active or 10-20 times less active than intact CS-1 in promoting the adhesion of lymphocytes expressing the resting form of the receptor. In cells which express the high avidity form of the receptor, LDV and CS-1 were equally effective in promoting cell adhesion and spreading. The avidity of the alpha 4 beta 1 complex could be altered with mAbs to beta 1 which specifically activate beta 1 dependent function. The high avidity form of the alpha 4 beta 1 complex could be induced on U937 cells, T, and B lymphoblastoid cell lines, or PHA-stimulated T cell blasts. Resting PBL could not be induced to bind LDV peptide conjugates by activating antibodies to beta 1 implying that two signals are required for LDV recognition by T cells. In conclusion, these data show clearly that the minimal peptide for the alpha 4 beta 1 complex in CS-1 is the LDV sequence. Although numerous cell populations can interact with intact CS-1 only cells which express an active alpha 4 beta 1 complex can bind the LDV sequence. This implies that cell interaction with the carboxy terminal cell-binding domain of fibronectin can be regulated at several levels: (a) alpha 4 beta 1 expression; (b) activation of the alpha 4 beta 1 complex; and (c) alternate splicing of CS-1 into V+ isoforms of fibronectin.     

Activation of the complement system during immunotherapy with recombinant IL-2. Relation to the development of side effects

Therapy with high doses of rIL-2 is complicated by the occurrence of hypotensive reactions and the development of a vascular leakage syndrome (VLS). In four patients, who together received seven cycles of high doses of IL-2 (up to 12 x 10(6) U per m2 per day), and who developed these side effects, we observed an unexpected increase in plasma levels of C3a, indicating activation of the complement system. C3a levels markedly increased during IL-2 therapy from 4 nmol/liter (mean level) before therapy to 23 nmol/liter at the end of the cycle. Activation of C3 occurred via the classical pathway inasmuch as C4a levels also increased during therapy. Mean daily C3a levels correlated with signs of the VLS, such as daily weight gain (p less than 0.001) and albumin levels (inverse correlation, p less than 0.001). In five additional patients, who together received seven cycles of lower doses of IL-2 (2 x 10(6) U per m2 per day) and who did not develop a VLS, only moderate increases in C3a levels (up to 13 nmol/liter) were observed. The highest levels at the first day of the regimen (mean: 7 nmol/liter) occurred 8 h after the IL-2 infusion. Thus, administration of IL-2 induces a dose-dependent activation of the complement system in vivo, which appeared to be related to the development of side effects of this therapy, such as the VLS.     

Structure of ricin A-chain at 2.5 A

Ricin has been refined in a crystallographic sense to 2.5 A resolution and the model for the A-chain (RTA) is described in detail. Because RTA is the first member of the class of plant toxins to be analyzed, this model probably defines the major structural characteristics of the entire family of these medically important proteins. Explanations are provided to rationalize amino acids that are conserved between RTA and a number of homologous plant and bacterial toxins. Eight invariant residues appear to be involved in creating or stabilizing the active site. In the active site Arg180 and Glu177 are hydrogen bonded to each other and also coordinate a water molecule; each of these groups may be important in the N-glycosidation reaction. Several other polar residues may play lesser roles in the mechanism, including tyrosines 80 and 123 and asparagines 78 and 209. A number of conserved hydrophobic residues are seen to cluster within several patches and probably drive the overall folding of the toxin molecule.     

Resveratrol prevents endothelial cells injury in high-dose interleukin-2 therapy against melanoma

Immunotherapy with high-dose interleukin-2 (HDIL-2) is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS). In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSC) and FoxP3(+)CD4(+) regulatory T cells (Treg). We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory T cell in the development of VLS.     

Design of targeted B cell killing agents

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.     

Antiapoptotic action of carbon monoxide on cultured vascular smooth muscle cells

Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) from the degradation of heme by the enzyme heme oxygenase. Because recent studies indicate that CO influences the properties of vascular SMCs, we examined whether this diatomic gas regulates apoptosis in vascular SMCs. Treatment of cultured rat aortic SMCs with a cytokine cocktail consisting of interleukin-1beta (5 ng/ml), tumor necrosis factor-alpha (20 ng/ml), and interferon-gamma (200 U/ml) for 48 hr stimulated apoptosis, as demonstrated by DNA laddering, caspase-3 activation, and annexin V staining. However, the exogenous addition of CO (200 ppm) completely blocked cytokine-mediated apoptosis. The antiapoptotic action of CO was partially reversed by the soluble guanylate cyclase inhibitor, H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM). In contrast, the p38 mitogen-activated protein kinase inhibitor, SB203580 (10 microM), had no effect on SMC apoptosis. These findings indicate that CO is a potent inhibitor of vascular SMC apoptosis and that it blocks apoptosis, in part, by activating the cGMP signaling pathway. The ability of CO to inhibit vascular SMC apoptosis may play a critical role in attenuating lesion formation at sites of arterial damage.     

NK cells and polymorphonuclear neutrophils are both critical for IL-2-induced pulmonary vascular leak syndrome

The mechanism of IL-2-induced vascular leak syndrome (VLS) is still poorly understood. Cells of both innate and adaptive immune systems have been implicated, but no definitive conclusions have been reached concerning their respective roles. In this study we report a new mouse model of IL-2-induced pulmonary VLS used to obtain a detailed analysis of the early events (sequestration of polymorphonuclear neutrophils and bronchoconstriction) and late events (modifications in the cell and protein content of bronchoalveolar lavages, followed by edema) that characterize this lung injury. This model and knockout animals are used to reconsider the importance of the different leukocyte lineages in early and late events. Recombinase-activating gene 2(-/-) mice are used to demonstrate that adaptive lymphocytes, including NK T cells, are not required for pulmonary VLS induction. By contrast, results obtained with newly described recombinase-activating gene 2(-/-)/IL-15(-/-) mice indicate that NK cells play a key role in both early and late events. In parallel, polymorphonuclear neutrophil depletion is used to evaluate the contributions made by these cells to the late alterations occurring in the lung. Furthermore, when used in combination with inhibition of NO synthase, granulocyte depletion was completely effective in protecting mice from the late events of IL-2-induced pulmonary VLS. Together our results indicate that both NK and PMN cells play a central role in the late events of IL-2-induced VLS.     

Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2

Adoptive immunotherapy with lymphokine-activated killer cells and recombinant interleukin 2 (IL 2) can produce significant reduction of visceral metastases in tumor-bearing mice and, as shown recently, in humans with disseminated cancer. Because further dose escalations of IL 2 have been prevented by the development of a vascular leak syndrome (VLS) in both mice and humans, we investigated this VLS in mice undergoing the systemic administration of high-dose IL 2. A model for quantitating capillary permeability was used in which 125I-bovine serum albumin was injected i.v., and 2 hr later, tissues were counted in a gamma analyzer. A permeability index (PI) was calculated by dividing the mean counts per minute (cpm) of tissues from IL 2-treated mice by those from control animals. The injection of IL 2 produced increases in vascular permeability that were most pronounced in the thymus, spleen, lungs, liver, and kidneys (PI = 18.0, 10.0, 9.7, 6.7, and 6.3, respectively, on day 6). The development of the VLS was highly dependent on the number of days of IL 2 treatment (for example, the lungs contained 638, 1382, 3350, and 6187 cpm after 0, 1, 3, and 6 days of IL 2, respectively). Moreover, the degree of the VLS was directly related to the dose of IL 2 administered. Measurement of the wet and dry weights of lungs from IL 2-treated mice demonstrated that IL 2 produced a dramatic increase in their water weight (from 0.10 g at base line to 0.22 g after 200,000 U of IL 2 for 6 days). The injection of the IL 2 excipient failed to induce capillary leakage in tissues. Immunosuppression of mice by pretreatment irradiation (500 rad) or by injection of cyclophosphamide or by concurrent use of cortisone acetate markedly reduced or eliminated the development of the VLS. Similarly, the VLS was not observed in nude mice receiving IL 2. Thus, the administration of IL 2 produces a dose-limiting VLS that may be mediated, directly or indirectly, by host lymphoid elements.     

Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.