Evidence for differential effects of sulphasalazine on systemic and mucosal immunity in rheumatoid arthritis

OBJECTIVE: To study the effects of sulphasalazine (SASP) on the systemic and mucosal humoral immune systems in patients with rheumatoid arthritis (RA). METHODS: Serum concentrations of interleukin 6 (IL-6), class and subclass specific IgG, IgA and IgM, IgA and IgG antigliadin antibodies and rheumatoid factors (RF) of IgG, IgA (including IgA1 and IgA2 subclasses) and IgM isotypes were measured before and 16 weeks after sulphasalazine (SASP) therapy in 15 female and three male patients with RA. Amounts of immunoglobulins in saliva and jejunal fluid were measured as estimates of mucosal humoral immunity. RESULTS: Serum concentrations of IgA and IgG decreased significantly during SASP therapy and correlated with reduced concentrations of IL-6. In addition, levels of circulating IgA RF, IgA anti-gliadin antibodies and IgM RF decreased significantly after the treatment. In contrast, immunoglobulin levels in saliva and jejunal fluid were unaltered. CONCLUSION: SASP exerts powerful but selective inhibitory effects on systemic immunoglobulin production, whereas no effects on mucosal immunoglobulin production were observed. The decreased systemic B cell activity may be mediated by downregulation of the production of IL-6, a cytokine with Ig switching properties.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Mechanisms for induction of acquired host immunity by neutrophil peptide defensins

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.     

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.     

Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.     

Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-gamma secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-gamma and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-gamma secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.     

A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.     

Mucosal antibody expression following rapid SIV(Mne) dissemination in intrarectally infected Macaca nemestrina

The early kinetics of antibody expression following transmucosal infection by SIV(Mne) were examined in several mucosal compartments in Macaca nemestrina. Five male-female pairs of macaques were inoculated intrarectally with SIV(Mne) E11S, a biological clone, and serially euthanized at 1, 2, 4, 8, and 12 weeks postinoculation. Plasma, tears, saliva, rectal secretions, and vaginal washes were collected serially and just prior to euthanasia. Both total and SIV-specific IgG and IgA levels were measured by immunoglobulin isotype-specific quantitative enzyme-linked immunosorbent assays (ELISAs), and were further examined by conventional and enhanced chemiluminescence (ECL) immunoblots. Virus coculture, polymerase chain reaction, and in situ hybridization assays revealed the systemic spread of virus as early as 1 week postinoculation in 8 of 10 animals. ECL immunoblots detected SIV-specific antibodies in mucosal samples collected 1 week postinoculation. The most dramatic increases in both total and SIV-specific IgA levels were detected in rectal secretion samples. In contrast, plasma and nonrectal mucosal samples from the same time points increased only slightly, suggesting that the most robust antibody response occurred at the portal of infection. Our results show that the SIV-infected macaque is an excellent model for studies designed to assess mucosal immune responses to primate lentivirus infections. Additional studies will assess the correlation between the antiviral protection afforded by candidate vaccines and mucosal antibody responses.     

Systemic and mucosal IgA responses to systemic antigen challenge in IgA nephropathy

IgA nephropathy (IgAN) is characterized by the deposition of glomerular IgA. The source of the deposited IgA is not known, but both the mucosal and systemic IgA systems have been implicated. In order to investigate mucosal and systemic antibody production to systemic antigen challenge in IgAN, 20 patients and 20 controls where immunized with tetanus toxoid (TT). While patients with IgAN responded with a similar serum IgG, IgA, IgA1, and IgA2 antibody response to controls, they did, however, produce more IgA1 antibodies relative to IgA2 (P < 0.05). No salivary IgA antibody response was observed to systemic immunization in controls; however, there was a significant IgA response to TT in the saliva of patients with IgAN. IgA antibodies were produced in vitro by Epstein Barr virus (EBV)-transformed peripheral blood lymphocytes (PBLs) obtained from control blood only when taken shortly (1 or 2 weeks) after immunization. Patients with IgAN produced significantly more IgA anti-TT positive cultures than controls and for a longer period (P < 0.01) after immunization. In contrast, IgG anti-TT was produced in EBV-transformed cultures at all time points, but with no difference between IgAN and controls in the proportion of IgG producing cultures. These results demonstrate increased IgA antibody production in both the systemic and mucosal IgA systems following systemic immunization in IgAN and suggest an abnormal overlap between the two systems in IgAN.     

Antibodies and antibody-secreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.     

Interleukin-5 expressed by a recombinant virus vector enhances specific mucosal IgA responses in vivo

Several in vitro studies have shown that murine interleukin-5 (mIL-5) enhances IgA production by activated mucosal B cells. To date, however, there is no evidence that this factor significantly up-regulates mucosal IgA responses in vivo. Here, we show that expression of the gene for mIL-5 in a recombinant vaccinia virus vector markedly increases IgA responses to co-expressed heterologous antigen in the lungs of mice given intranasal inocula of the virus. The elevated local IgA responses to vectors expressing mIL-5 peaked at a fourfold higher level than those elicited by control virus at 14 days after infection and were sustained for at least 4 weeks. Increased IgA responses were abrogated in mice treated with monoclonal antibody against mIL-5 and were not detected in systemic lymphoid tissue. No enhancement of specific IgG levels was found either locally or systemically. Our results indicate that mIL-5 selectively enhances the development of mucosal IgA responses in vivo and suggest that expression of this factor in mucosal vaccine vectors may stimulate local immune reactivity.     

IL-12 is a potent neonatal vaccine adjuvant

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.     

Chronic active hepatitis induced by Helicobacter hepaticus in the A/JCr mouse is associated with a Th1 cell-mediated immune response

Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-gamma) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-gamma in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.     

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.     

Controlled lipidation and encapsulation of peptides as a useful approach to mucosal immunizations

To generate a useful strategy for mucosal immunization, we have developed an approach of lipidating a multiple Ag peptide (MAP) containing part of the V3 loop from HIV-1 gp120IIIB. In this work, we compare two delivery systems, lipidated MAP in PBS and encapsulation in poly(DL-lactide-co-glycolide) microparticles. Subcutaneous immunization, followed by intragastric administration of MAP peptide entrapped or not entrapped in microparticles, induced mucosal and systemic immune responses at local and distant sites, including mucosal IgA in saliva, vaginal secretions and feces, and IgG in blood. However, lipidated Ag delivered in microparticles induced higher levels of mucosal Abs, particularly of intestinal IgA, and generated CTL responses. In contrast, lipidated MAP delivered by nasal route microparticles was less effective in inducing CTL responses. These results demonstrate the feasibility of using a lipidated multimeric peptide for mucosal immunization to stimulate both systemic and mucosal immune systems, including the genital tract, irrespective of the route or method of delivery and without requiring the use of a carrier or an extraneous adjuvant.     

Interleukin-6 and small intestinal luminal immunoglobulins

Our aim was to determine the relationships between interleukin-6 and immunoglobulin levels within small intestinal luminal secretions. Twenty adult subjects with small intestinal bacterial overgrowth (N = 13), irritable bowel syndrome (N = 4), and nonulcer dyspepsia (N = 3) underwent endoscopic aspiration of secretions from the small intestinal mucosal surface for assessment of IL-6, IgA1, IgA2, IgM, IgG1, IgG2, IgG3, and IgG4 concentrations. Serum immunoglobulin concentrations and small intestinal histology were also determined. IgA2 and IgG3 were the predominant IgA and IgG subclasses in luminal secretions in 19/20 (95%) and 20/20 (100%) subjects, respectively. IgA1 and IgG1 predominated in serum in all subjects. No subject had villous atrophy. Luminal IL-6 concentrations correlated significantly with luminal IgA2, IgM, and IgG3 concentrations but not with IgA1 or any other IgG subclass levels. Conversely, luminal IL-6 or immunoglobulin concentrations did not correlate significantly with levels of any immunoglobulin isotype in serum. These observations suggest that important relationships exist between local IL-6 and IgA2, IgM, and IgG3 responses in human small intestinal luminal secretions. Local investigation is mandatory when assessing intestinal immune activity.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Kinetics of specific immunoglobulin A, M and G production in the duodenal and caecal mucosa of chickens infected with Eimeria acervulina or Eimeria tenella

The development and appearance of antibody was studied in the intestine and serum from histocompatible GB1 chickens orally infected with oocysts of Eimeria acervulina (restricted to the duodenum) or Eimeria tenella (restricted to the caeca). The local immune response was measured as the specific antibody levels in the supernatants of intestinal fragments (duodenum and caecum) maintained in culture for 16 h at 41 degrees C, 5% CO2, 95% air. Specific IgM was detected 1 week after E. acervulina infection, and the specific IgA and IgG contents of the duodenum and caecum were significantly elevated (P < 0.001) after 2 weeks. The intestinal specific IgG content was raised. E. tenella infection resulted in specific IgA only in the parasitized area during the second week post-infection (P < 0.05). Specific IgM and IgG were both detected in the duodenum and caecum, respectively, 1 and 2 weeks p.i. Production of parasite-specific immunoglobulins was always significantly higher in the parasitized than in the unparasitized areas (caeca for E. acervulina, duodenum for E. tenella). This ex vivo culture assay of intestinal fragments used to measure the mucosal immune response of intestinal areas showed a significant production of specific IgA and IgM. In addition, high levels of IgG were also measured. The role of this specific IgG in Eimeria infection remains to be determined.