Moving to evidence-based practice for pain management in the critical care setting.

Widespread application of EBPM by bedside providers is needed to demonstrate the success of pain management strategies on patient outcomes. This goal is not easy to attain and generally requires time, patience, and a multidisciplinary team approach. Implementation and evaluation of pain interventions increase awareness and knowledge of pain management strategies and can result in an overall improvement in pain management. The literature and guidelines recommend the use of specific strategies to ensure practice change. Studies suggest that a more intensive or "active" effort to alter practice is generally most successful. The pain management program should be marketed so that both the health care providers and patients are aware of the goal and resources available. It may take 3 to 5 years to infuse the change and see the improvement. Reinfusion over time also needs to be planned. Bedside practitioners need to have knowledge of the current best evidence in pain management of the critically ill patient. Barriers to implementation must be eliminated so that practitioners can conscientiously and judiciously implement strategies to relieve pain. Opinion leaders and change agents need to be available to continually champion EBPM, and prompts to ask about pain should be provided to practitioners and patients.

Effect of PDI overexpression on recombinant protein secretion in CHO cells.

In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges. Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL-15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). Secretion of TNFR:Fc (a disulfide-rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum. PDI overexpression did not result in intracellular retention of IL15. The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein.

Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways.

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-gamma or -alpha and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor (R)alpha(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-3Ralpha(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

Poxvirus genomes encode a secreted, soluble protein that preferentially inhibits beta chemokine activity yet lacks sequence homology to known chemokine receptors.

Poxvirus genomes encode several proteins which inhibit specific elements of the host immune response. We show the "35K" virulence gene in variola and cowpox viruses, whose vaccinia and Shope fibroma virus equivalents are strongly conserved in sequence, actually encodes a secreted soluble protein with high-affinity binding to virtually all known beta chemokines, but only weak or no affinity to the alpha and gamma classes. The viral protein completely inhibits the biological activity of monocyte chemotactic protein-1 (MCP-1) by competitive inhibition of chemokine binding to cellular receptors. As all beta chemokines are also shown to cross-compete with MCP1 binding to the viral protein, we conclude that this viral chemokine inhibitor (vCCI) not only interacts through a common binding site, but is likely a potent general inhibitor of beta chemokine activity. Unlike many poxvirus virulence genes to date, which are clearly altered forms of acquired cellular genes of the vertebrate immune system, this viral chemokine inhibitor (vCCI) shares no sequence homology with known proteins, including known cellular chemokine receptors, all of which are multiple membrane-spanning proteins. Thus, vCCI presumably has no cellular analogue and instead may be the product of unrelenting sequence variations which gave rise to a completely new protein with similar binding properties to native chemokine receptors. The proposed function of vCCI is inhibition of the proinflammatory (antiviral) activities of beta chemokines.

TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.

Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

Activation of nuclear transcription factor NF-kappaB by interleukin-1 is accompanied by casein kinase II-mediated phosphorylation of the p65 subunit.

In fibroblasts and hepatoma cells, interleukin-1 (IL-1) treatment results in the rapid nuclear accumulation of the transcription factor NF-kappaB, present largely as p65 (RelA)/p50 heterodimers. It is well established that this process is dependent in large part upon the phosphorylation and subsequent degradation of the cytosolic inhibitor IkappaB. We looked for other IL-1-induced modifications of NF-kappaB components and found that, in both cell types, IL-1 stimulation led, within minutes, to phosphorylation of both NF-kappaB p65 and p50. Phosphorylation of p65 was sustained for at least 30 min after addition of the cytokine and occurred principally upon serine residues. Immunoprecipitates of NF-kappaB complexes contained an associated protein kinase, the biochemical characteristics of which were indistinguishable from casein kinase II (CKII). Purified CKII efficiently phosphorylated p65 in vitro, apparently on the same major sites that became phosphorylated in intact IL-1-treated cells. Although IL-1 treatment caused little apparent stimulation of total cellular CKII activity, the fraction that was specifically associated with NF-kappaB complexes was markedly elevated by the cytokine. The association of CKII with NF-kappaB occurred in the cytoplasm, suggesting that this phosphorylation might be involved either in control of translocation of the activated complex or in modulation of its DNA binding properties.

Identification and characterization of a new member of the TNF family that induces apoptosis.

A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.

Characterization of B-cell lines established from two X-linked severe combined immunodeficiency patients: interleukin-15 binds to the B cells but is not internalized efficiently.

X-linked severe combined immunodeficiency (XSCID) is characterized by absent or profoundly reduced numbers of T cells and normal numbers of B cells in the circulation. Affected patients have mutations of the interleukin-2 (IL-2) receptor gamma chain gene. Using Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) established from two unrelated XSCID patients, we could show that neither expressed the IL-2 receptor gamma chain on the cell surface. A novel cytokine IL-15, which has biologic activities similar to those of IL-2, could bind to the XSCID B-LCLs in the absence of the gamma chain, although both the beta and gamma chains of the human IL-2 receptor were previously shown to be required for IL-15 binding by transfected COS cells. Furthermore, a significant reduction and delay of IL-15 internalization by B lymphoblasts from XSCID patients was observed when compared with that of normal control B-LCLs. These results show the existence of a novel IL-15-specific receptor component that contributes to IL-15 binding but is insufficient for IL-15 internalization in the absence of the IL-2 receptor gamma chain.

Fas ligand mediates activation-induced cell death in human T lymphocytes.

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.

The Mpl receptor (Mpl-R) is a cytokine receptor belonging to the hematopoietin receptor superfamily for which a ligand has been recently characterized. To study the lineage distribution of Mpl-R in normal hematopoietic cells, we developed a monoclonal antibody (designated M1 MoAb) by immunizing mice with a soluble form of the human Mpl-R protein. With few exceptions, Mpl-R was detected by indirect immunofluorescent analysis on all human leukemic hematopoietic cell lines with pluripotential and megakaryocytic phenotypes, but not on other cell lines. By immunoprecipitation and immunoblotting, M1 MoAb recognized a band at 82 to 84 kD corresponding to the expected size of the glycosylated receptor. Among normal hematopoietic cells, M1 MoAb strongly stained megakaryocytes (MK) and Mpl-R was detected on platelets by indirect immunofluorescence staining or immunoblotting. On purified CD34+ cells, less than 2% of the population was stained, but the labeling was weak and just above the threshold of detection. However, dual-labeling with the M1 and antiplatelet glycoprotein MoAbs showed that most Mpl-R+/CD34+ cells coexpressed CD41a, CD61, or CD42a, suggesting that cell surface appearance of Mpl-R and platelet glycoproteins could be coordinated. M1-positive and M1-negative subsets were sorted from purified CD34+ cell populations. Colony assays showed that the absolute number of hematopoietic progenitors was extremely low and no primitive progenitors were present in the CD34+/Mpl-R+ fraction. However, this cell fraction was significantly enriched in low proliferative colony-forming units-MK. When the CD34+/Mpl-R+ fraction was grown in liquid culture containing human aplastic serum and a combination of growth factors, mature MK were seen as early as day 4, whereas the predominant cell population was erythroblasts on day 8. Similar data were also obtained with the CD34+/Mpl-R- fraction with, however, a delay in the time of appearance of both MK and erythroblasts. In conclusion, Mpl-R is a cytokine receptor restricted to the MK cell lineage. Its expression is low on CD34+ cells and these cells mainly correspond to late MK progenitors and transitional cells. These data indicate that the action of the Mpl-R ligand might predominate during the late stages of human MK differentiation.

Regulation of apoptosis and T cell activation by Fas-specific mAb.

Fas was initially described as a molecule expressed on the surface of certain cell lines that could mediate programmed cell death (apoptosis) subsequent to ligation by specific mAb. To determine whether mAb to other epitopes on the Fas molecule might mediate other functions, we generated a panel of mAb to the extracellular portion of human Fas. Significant lysis of Fas-expressing target cells was only observed when the new mAb were first bound to a solid-phase support and not when the mAb were added in solution. However, several of these mAb inhibited the killing of target cells induced by the prototypic Fas-specific mAb, CH-11. Those mAb that inhibited apoptosis of target cells mediated by the CH-11 mAb also blocked lysis of target cells mediated by cells expressing Fas ligand. Finally, some of the Fas-specific mAb were found to co-stimulate proliferation of peripheral blood T cells in the presence of immobilized CD3 mAb. Thus, the data indicate the existence of a complex set of interactions mediated by Fas in both normal and transformed lymphoid cells that may have important implications regarding the role(s) of this molecule in regulation of immune responses.

Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is able to promote the growth of mouse primordial germ cells (PGCs) in culture. It is unclear whether LIF acts directly on PGCs or indirectly via feeder cells or embryonic somatic cells. To understand the role of LIF in PGC growth, we have carried out molecular and cell culture analyses to investigate the role of both the LIF ligand and its receptor in PGC development. LIF is able to stimulate PGC growth independently of the presence of feeder cells supporting the hypothesis that LIF acts directly on PGCs to promote their growth. We show here that transcripts for the low-affinity LIF receptor (LIFR), an integral component of the functional LIF receptor complex, are expressed in the developing gonad. Fluorescence-activated cell sorter (FACS) analysis, using an anti-LIFR antiserum, demonstrates that LIFR is present on the surface of PGCs, suggesting that PGCs are likely to be a direct target of LIF action in culture. Signalling via LIFR is essential for PGC growth in culture since the anti-LIFR antiserum, which blocks LIF binding to its receptor, abolishes PGC survival in culture. Two LIF-related cytokines, namely oncostatin M and ciliary neurotrophic factor, can also promote PGC growth in culture in addition to LIF. Thus one or more of these LIFR-dependent cytokines may play an important role in PGC development in mice.

Fas transduces activation signals in normal human T lymphocytes.

The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.

Identification of a distinct low-affinity receptor for human interleukin-4 on pre-B cells.

Biotinylated interleukin-4 (IL-4) was used to examine IL-4 receptor (IL-4R) expression on a range of human B-cell lines by flow cytometry. Using high concentrations of biotinylated IL-4, we have identified a novel low-affinity IL-4 receptor expressed at high levels on pre-B lines. Expression of this low-affinity receptor did not correlate with detected mRNA levels for the previously cloned receptor or with reactivity of two anti-human IL-4R monoclonal antibodies (MoAb). Radiolabeled IL-4 cross-linking studies using pre-B lines showed a doublet of 65 to 75 Kd in contrast to the 110- to 130-Kd molecule detected on cells expressing the cloned IL-4R. A soluble IL-4 binding protein (IL-4bp) was purified from the supernatants of three pre-B lines expressing the low-affinity receptor on their surface. IL-4bp could block both IL-4-mediated CD23 induction on tonsil B cells and IL-4-induced inhibition of proliferation of the pre-B line JM1. Partial N-terminal amino acid sequence was obtained from purified IL-4bp that confirmed this protein to be novel. A 12 amino acid peptide based on the IL-4bp sequence was used to produce a polyclonal antiserum that was reactive with purified IL-4bp, and also bound to the surface of pre-B cells but not to murine CTLL cells transfected with the human IL-4R. Blocking MoAb against the previously characterized high-affinity receptor inhibited IL-4-mediated proliferation of hIL-4R+ CTLL cells but had no effect on IL-4-induced inhibition of JM1 cell proliferation, and only partially inhibited IL-4-mediated CD23 and sIgM induction and proliferation of tonsil B cells. The data presented here provide evidence for a novel cell-surface expressed low-affinity IL-4R that also exists as a biologically active soluble IL-4 binding protein.

Molecular cloning and characterization of a novel receptor protein tyrosine kinase from human placenta.

Using a polymerase chain reaction-based approach we have isolated and characterized a cDNA (HPK-6) from human placental RNA encoding a novel receptor protein tyrosine kinase. This receptor tyrosine kinase has a unique extracellular domain, with an immunoglobulin-like domain at the amino terminus followed by three EGF-like cysteine repeats and three fibronectin type III repeats, giving the HPK-6 gene extracellular domain a novel combination of structural motifs. A comparison of the HPK-6 sequence with other receptor tyrosine kinases shows that the HPK-6 gene is the human homolog of the murine tek gene and very closely related to the recently described receptor tyrosine kinase tie. The HPK-6 gene is expressed predominantly in placenta and lung, with a lower level in umbilical vein endothelial cells, brain and kidney. The HPK-6 cDNA, when transfected into COS-7 cells, encodes a 140-kDa protein with in vitro kinase activity.

Independent binding of interleukin-1 alpha and interleukin-1 beta to type I and type II interleukin-1 receptors.

Interleukin (IL)-1 refers to a group of three polypeptide hormones with a wide range of cellular targets. Two types of IL-1 receptor have been identified and characterized by cDNA cloning. Both human type I and type II IL-1 receptors contain extracellular domains of approximately 310 residues and a single membrane-spanning region. The type I receptor contains a cytoplasmic domain of 213 residues. The cytoplasmic region of the type II receptor is 29 residues in length. It has been found recently that a number of cells express both forms of receptor. By analogy with other cytokine receptor systems, the two IL-1 receptors might be expected to form a heterodimeric complex, the type II receptor being an alpha-chain-like structure, functioning only to bind ligand, and associating with the type I receptor (a beta-chain-like structure) which would transduce signals. In this report we show that this is not the case, but rather that IL-1, when complexed to type II receptor, cannot bind type I receptors, and vice versa. These data show that the complex patterns often observed for IL-1 binding to cells cannot be accounted for by the same type of mechanism that underlies the behavior of, for example, the IL-2 system.

Regulation of murine in vivo IgG and IgE responses by a monoclonal anti-IL-4 receptor antibody.

Although the cytokine interleukin 4 (IL-4) stimulates LPS-activated mouse B lymphocytes to secrete both IgG1 and IgE, an anti-IL-4 antibody completely inhibits IgE responses but has little or no effect on several in vivo IgG responses. IL-4 might, therefore, have a restricted role in the generation of in vivo humoral immune responses. Alternatively, IgG1 responses might be stimulated by IL-4 secreted by T cells that are interacting directly with B cells, so that anti-IL-4 antibody cannot neutralize IL-4 before it binds to a B cell IL-4 receptor. In contrast, an antibody that blocks the IL-4 receptor (IL-4R) should equally inhibit responses to IL-4 produced proximal to or distant from a B cell. This reasoning led us to determine the ability of an anti-IL-4R mAb to affect antibody production in mice injected with a goat antibody to mouse IgD (GaM delta) or inoculated with the nematode parasite Heligmosomoides polygyrus. Anti-IL-4R mAb, like anti-IL-4 mAb, blocked IgE responses by greater than 95% and enhanced IgG2a responses to a variable extent. Anti-IL-4R mAb, however, had only a modest and variable inhibitory effect on the induction of IgG1 responses, although it caused these responses to terminate more rapidly. A combination of anti-IL-4 and anti-IL-4R mAbs totally blocked goat anti-mouse IgD antibody (GaM delta)-induced IgE production but had no additive inhibitory effect on IgG1 production. These observations are most consistent with the view that IL-4 is required for a primary IgE response, but has relatively little role in the induction of IgG1 responses in the in vivo systems studied.