IgA production without mu or delta chain expression in developing B cells.

Surface, membrane-bound, immunoglobulin M (IgM) or IgD expression early in B cell ontogeny is considered essential for the differentiation of antibody-producing cells in mammals; only in IgM+ B cells is the heavy chain locus rearranged to express antibodies of other classes. We show here that IgA is selectively expressed in muMT mice, which lack IgM or IgD expression and have a pro-B cell developmental block. muMT IgA binds proteins of commensal intestinal bacteria and is weakly induced by Salmonella infection, although not through conventional immunization. This muMT IgA pathway requires extrasplenic peripheral lymphoid tissues and may be an evolutionarily primitive system in which immature B cells switch to IgA production at peripheral sites.

Measurement of mouse and human interleukin 5.

Two fundamentally different types of assays are available for quantitating interleukin 5 in biological samples. One type, a bioassay, is based on the ability of IL-5 to enhance B cell proliferation and immunoglobulin secretion or eosinophil proliferation and differentiation. The other type of assay, an ELISA, uses antibodies against IL-5 to capture and quantitate IL-5 in samples. Advantages and disadvantages of each assay are discussed. The bioassay described in this unit, utilizing BCL(1) cells as the indicator line, has been designed primarily to assay murine IL-5; however, it can also be used to measure human IL-5. The IL-5 ELISA, while sensitive and specific, provides no information about biological activity.

Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group.

BACKGROUND: Neutralization of tumor necrosis factor a (TNF-alpha) for three to six months reduces the symptoms and signs of rheumatoid arthritis. However, the capacity of this approach to effect a more sustained benefit and its effect on joint damage are not known. METHODS: We treated 428 patients who had active rheumatoid arthritis despite methotrexate therapy with placebo or infliximab, a chimeric monoclonal antibody against TNF-alpha, in intravenous doses of 3 or 10 mg per kilogram of body weight every 4 or 8 weeks in combination with oral methotrexate for 54 weeks. We assessed clinical responses with use of the criteria of the American College of Rheumatology, the quality of life with a health-status questionnaire, and the effect on joint damage radiographically. RESULTS: The combination of infliximab and methotrexate was well tolerated and resulted in a sustained reduction in the symptoms and signs of rheumatoid arthritis that was significantly greater than the reduction associated with methotrexate therapy alone (clinical response, 51.8 percent vs. 17.0 percent; P<0.001). The quality of life was also significantly better with infliximab plus methotrexate than with methotrexate alone. Radiographic evidence of joint damage increased in the group given methotrexate, but not in the groups given infliximab and methotrexate (mean change in radiographic score, 7.0 vs. 0.6, P<0.001). Radiographic evidence of progression of joint damage was absent in infliximab-treated patients whether or not they had a clinical response. CONCLUSIONS: In patients with persistently active rheumatoid arthritis despite methotrexate therapy, repeated doses of infliximab in combination with methotrexate provided clinical benefit and halted the progression of joint damage.

Immune activation in the intestinal mucosa before the onset of colitis in Galphai2-deficient mice.

G-protein subunit Galphai2-deficient mice spontaneously develop an inflammatory bowel disease that clinically and histopathologically resembles ulcerative colitis in humans. The aim of this study was to determine whether immunological changes precede the development of colitis in Galphai2-deficient mice. Therefore, Galphai2-deficient mice with no clinical or histopathological signs of colitis were compared with Galphai2-deficient mice with established colitis and wild-type animals, concerning immunological parameters. Healthy Galphai2-deficient mice displayed an increased frequency of CD4+ T cells and a decreased frequency of CD19+ B lymphocytes in the intestinal mucosa compared with control mice. The CD4+ population was characterized by a memory phenotype, i.e. increased expression of CD44 and decreased expression of CD45RB and CD62L, as well as increased expression of the mucosal homing receptors integrins alpha4beta7 and alphaEbeta7. Production of pro-inflammatory cytokines, interleukin (IL)-1beta and interferon (IFN)-gamma, were increased in Galphai2-deficient mice before clinical signs of disease were evident. In addition, total immunoglobulin (Ig)G and IgA levels in large intestinal secretions were increased significantly compared with wild-type mice, and antibodies specific for the normal intestinal flora in large intestinal secretions were present in Galphai2-deficient mice several weeks before the onset of colitis. In contrast, antibodies against tropomyosin, a putative autoantigen in human ulcerative colitis, were not found in Galphai2-deficient mice before the onset of colitis, although they were present in animals with established disease. In conclusion, activation of the intestinal immune system precedes histopathological and clinical signs of inflammation in Galphai2-deficient mice, suggesting that immune abnormalities play an important role in the induction of colitis.

Cell adhesion antagonists: synthesis and evaluation of a novel series of phenylalanine based inhibitors.

Several phenylalanine based inhibitors were synthesized as antagonists of the leukocyte cell adhesion process that is mediated through the interactions of the mucosal addressin cell adhesion molecule (MAdCAM) and the integrin alpha4beta7. Analogues 20, 21, 22 and 24 displayed inhibition of adhesion in a cell based assay in the low micromolar range.

A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria.

The immunoglobulin A (IgA) is produced to defend mucosal surfaces from environmental organisms, but host defenses against the very heavy load of intestinal commensal microorganisms are poorly understood. The IgA against intestinal commensal bacterial antigens was analyzed; it was not simply "natural antibody" but was specifically induced and responded to antigenic changes within an established gut flora. In contrast to IgA responses against exotoxins, a significant proportion of this specific anti-commensal IgA induction was through a pathway that was independent of T cell help and of follicular lymphoid tissue organization, which may reflect an evolutionarily primitive form of specific immune defense.

Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A.

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4(+) T cells or CD8(+) T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens.

Mucosal immunity to influenza without IgA: an IgA knockout mouse model.

IgA knockout mice (IgA-/-) were generated by gene targeting and were used to determine the role of IgA in protection against mucosal infection by influenza and the value of immunization for preferential induction of secretory IgA. Aerosol challenge of naive IgA-/- mice and their wild-type IgA+/+ littermates with sublethal and lethal doses of influenza virus resulted in similar levels of pulmonary virus infection and mortality. Intranasal and i.p. immunization with influenza vaccine plus cholera toxin/cholera toxin B induced significant mucosal and serum influenza hemagglutinin-specific IgA Abs in IgA+/+ (but not IgA-/-) mice as well as IgG and IgM Abs in both IgA-/- and IgA+/+ mice; both exhibited similar levels of pulmonary and nasal virus replication and mortality following a lethal influenza virus challenge. Monoclonal anti-hemagglutinin IgG1, IgG2a, IgM, and polymeric IgA Abs were equally effective in preventing influenza virus infection in IgA-/- mice. These results indicate that IgA is not required for prevention of influenza virus infection and disease. Indeed, while mucosal immunization for selective induction of IgA against influenza may constitute a useful approach for control of influenza and other respiratory viral infections, strategies that stimulate other Igs in addition may be more desirable.

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes.

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.

Ialpha exon-replacement mice synthesize a spliced HPRT-C(alpha) transcript which may explain their ability to switch to IgA. Inhibition of switching to IgG in these mice.

Antibody class switching is regulated by transcription of unrearranged C(H) genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that CH gene, the Ialpha exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the Ialpha exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C(alpha) gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal alpha GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig alpha locus may interfere with interaction of the promoters for gamma GL transcripts with the 3' IgH enhancer.

Antibody-independent protective mucosal immunity to gastric helicobacter infection in mice.

Helicobacter pylori infection of the gastric mucosa can result in gastritis and peptic ulcer disease. Although vaccination can induce protective immunity in animal models of Helicobacter infection, the mechanism(s) of protective immunity has not been fully elucidated. This study was designed to determine whether humoral immune responses are required for protective Helicobacter immunity. IgA-deficient or immunoglobulin-deficient mice were orally immunized against Helicobacter felis and then challenged with live H. felis. Both groups were protected at levels comparable to that of wild-type mice. Additionally, inflammation was equivalent in extent and character between wild-type and antibody-deficient mice. Therefore antibody-independent mechanisms of immunity can protect mice against gastric Helicobacter infection.

Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. ATTRACT Study Group.

BACKGROUND: Not all patients with rheumatoid arthritis can tolerate or respond to methotrexate, a standard treatment for this disease. There is evidence that antitumour necrosis factor alpha (TNFalpha) is efficacious in relief of signs and symptoms. We therefore investigated whether infliximab, a chimeric human-mouse anti-TNFalpha monoclonal antibody would provide additional clinical benefit to patients who had active rheumatoid arthritis despite receiving methotrexate. METHODS: In an international double-blind placebo-controlled phase III clinical trial, 428 patients who had active rheumatoid arthritis, who had received continuous methotrexate for at least 3 months and at a stable dose for at least 4 weeks, were randomised to placebo (n=88) or one of four regimens of infliximab at weeks 0, 2, and 6. Additional infusions of the same dose were given every 4 or 8 weeks thereafter on a background of a stable dose of methotrexate (median 15 mg/week for > or =6 months, range 10-35 mg/wk). Patients were assessed every 4 weeks for 30 weeks. FINDINGS: At 30 weeks, the American College of Rheumatology (20) response criteria, representing a 20% improvement from baseline, were achieved in 53, 50, 58, and 52% of patients receiving 3 mg/kg every 4 or 8 weeks or 10 mg/kg every 4 or 8 weeks, respectively, compared with 20% of patients receiving placebo plus methotrexate (p<0.001 for each of the four infliximab regimens vs placebo). A 50% improvement was achieved in 29, 27, 26, and 31% of infliximab plus methotrexate in the same treatment groups, compared with 5% of patients on placebo plus methotrexate (p<0.001). Infliximab was well-tolerated; withdrawals for adverse events as well as the occurrence of serious adverse events or serious infections did not exceed those in the placebo group. INTERPRETATION: During 30 weeks, treatment with infliximab plus methotrexate was more efficacious than methotrexate alone in patients with active rheumatoid arthritis not previously responding to methotrexate.

Immunity to vaginal HSV-2 infection in immunoglobulin A knockout mice.

An immunoglobulin A (IgA) knockout (KO) mouse was used to study the role of IgA in protective immunity against vaginal infection with herpes simplex virus-type 2 (HSV-2). Intact and KO mice were immunized intravaginally (IVAG) with attenuated HSV-2, challenged IVAG with wild-type virus 6 weeks later and evaluated for vaginal infection and neurological disease. Non-immunized/challenged intact and KO mice showed vaginal infection and succumbed to neurological disease, while immunized/challenged mice exhibited reduced or no vaginal infection and no neurological disease. Log 2.5 enzyme-linked immunoassay (ELISA) titres of viral IgA, immunoglobulin G (IgG) and immunoglobulin M (IgM) in vaginal secretions collected from intact immune mice before challenge were 0.6+/-0.3, 6.4+/-0.32 and 0.0, while those in KO immune mice were 0.0, 6.7+/-0.19 and 3.0+/-0.29, respectively. Twenty-four hours after challenge, the percentage of vaginal epithelium that was infected in non-immune intact and KO mice was 2.0+/-0.6 and 2.4+/-0.6, which was reduced to 0.2+/-0.1 and 0.1+/-0.06 in immune intact and KO mice, respectively. No shed virus protein was detected in vaginal secretions 3 days after challenge in any immune mouse, whereas titres were 1400 and 1700 in the two groups of non-immune mice. Thus, immune protection against vaginal HSV-2 infection was similar in both KO and intact mice, indicating that this mucosal immunity does not depend mainly on IgA.

G(alpha)i2-deficient mice with colitis exhibit a local increase in memory CD4+ T cells and proinflammatory Th1-type cytokines.

Mice with targeted deletion of the G protein G(alpha)i2 develop an inflammatory bowel disease closely resembling ulcerative colitis. To better define disease pathogenesis, the mucosal immune system in G(alpha)i2-deficient mice was studied. Phenotypic analysis of large intestine lamina propria lymphocytes revealed a large increase in memory CD4+ T cells (CD44high, CD45RBlow, CD62Llow). Furthermore, expression of the mucosal homing receptor integrin beta7 was increased on mucosal, but not systemic, CD4+ T cells. Analysis of cytokine production revealed a marked increase in proinflammatory Th1-type cytokines in inflamed colons, as compared with wild-type mice or G(alpha)i2-deficient mice without colitis. Thus, IFN-gamma and IL-1beta levels were increased 13-fold and 30-fold, respectively, with more modest increases in IL-6 levels (5-fold) and TNF levels (2-fold). Inflamed colons of G(alpha)i2-deficient mice also demonstrated increased IL-12 p40 mRNA levels. No increase in IL-2, IL-4, IL-5, and IL-10 was seen. Large intestinal epithelial cells in G(alpha)i2-deficient mice with colitis were found by immunohistochemistry to express increased levels of both MHC class I and class II Ags. Colitis was associated with increased IgG levels (60-fold increase), predominantly IgG2a (135-fold increase), in large but not small intestinal secretions. This was shown by ELISPOT analysis to result from local production within the lamina propria.

Differential activation requirements of isotype-switched B cells.

In the present studies, we compared the activation requirements of sIgM+/sIgD+ B cells with those of isotype-switched sIgM-/sIgA+ B cells. We found that whereas sIgM+ B cells respond to T cell-independent (TI) and T cell-dependent (TD) Ag with no significant bias toward one stimulus, sIgA+ B cells were deficient in their ability to respond to antigen receptor cross-linking but responded remarkably well to TD stimuli. Thus, dextran-conjugated anti-IgA antibody (anti-IgA-dextran), anti-kappa-dextran, or various immobilized anti-IgA antibodies (Ab) induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA+ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand-bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 10(5) cells). This pattern of sIgA+ B cell responsiveness was noted with both germinal center peanut agglutininhi (PNAhi) and non-germinal center PNAlo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA+ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%-94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype-switched B cells.

IgA class switch in I alpha exon-deficient mice. Role of germline transcription in class switch recombination.

Studies have implicated defective Ig class switch in the pathogenesis of IgA deficiency. To understand better the molecular events that regulate IgA class switch, a 1.4-kb region of the IgA locus containing the I alpha exon was replaced with a human hypoxanthine phosphoribosyltransferase minigene by gene targeting in murine embryonic stem cells. The I alpha exon-deficient mice derived from these embryonic stem cells had normal IgA levels in serum and secretions and normal numbers of IgA B cells in Peyer's patches and spleen. Further, I alpha exon-deficient B cells efficiently underwent IgA class switch in vitro, despite the absence of I alpha exon-containing germline transcripts. Notably, I alpha exon-deficient B cells did not require TGF-beta for IgA class switch since stimulation with LPS alone led to IgA expression. Nonetheless, whereas I alpha exon-deficient B cells constitutively expressed human hypoxanthine phosphoribosyltransferase transcripts, they did not produce IgA in the absence of LPS stimulation. These results demonstrate that the I alpha exon or transcripts containing the I alpha exon are not required for IgA class switch. Further, the effects of TGF-beta on I alpha locus transcription can be supplanted by expression of a heterologous minigene at that locus, but a second signal is required for the induction of IgA class switch.

Gi2 alpha protein deficiency: a model of inflammatory bowel disease.

Mice deficient for the G protein subunit Gi2 alpha were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 Gi2 alpha-deficient mice with colitis also developed adenocarcinomas of the colon. Gi2 alpha-deficient thymocytes displayed two- to fourfold increases in mature CD4+8- and CD4-8+ phenotypes, an approximately threefold increase in high-intensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of Gi 2 alpha-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumour necrosis factor, and interferon-gamma production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that Gi 2 alpha-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies.

Ulcerative colitis and adenocarcinoma of the colon in G alpha i2-deficient mice.

G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.