Generation of a complement-independent bactericidal IgM against a relapsing fever Borrelia

The spirochetemia of relapsing fever in mice is cleared by a complement-independent, polyclonal IgM response with reactivity to two prominent Ags of 20 and 35 kDa. In this study, we have dissected the polyclonal IgM Ab response against a relapsing fever spirochete to determine the specificity of its complement-independent bactericidal properties. Our experimental approach selectively generated an IgM murine mAb from the early specific immune response to a variable outer membrane protein. This IgM is bactericidal in the absence of complement and is part of the polyclonal Ab response that mediates the clearance of this bacterium from the blood. Purified monoclonal IgM caused direct structural damage to the outer membrane of the spirochete, in the absence of complement, and protected both B cell- and C5-deficient mice from challenge when administered passively. The direct, complement-independent, bactericidal activity of Abs is a critical mechanism of host defense against infection.     

IL-10 helps control pathogen load during high-level bacteremia

During relapsing fever borreliosis, a high pathogen load in the blood occurs at times of peak bacteremia. Specific IgM Abs are responsible for spirochetal clearance so in absence of B cells there is persistent high-level bacteremia. Previously, we showed that B cell-deficient mice persistently infected with Borrelia turicatae produce high levels of IL-10 and that exogenous IL-10 reduces bacteremia. This suggested that IL-10 helps reduce bacteremia at times of high pathogen load by a B cell-independent mechanism, most likely involving innate immunity. To investigate this possibility, we compared B. turicatae infection in RAG2/IL-10(-/-) and RAG2(-/-) mice. The results showed that IL-10 deficiency resulted in significantly higher bacteremia, higher TNF levels, and early mortality. Examination of the spleen and peripheral blood showed markedly increased apoptosis of immune cells in infected RAG2/IL-10(-/-) mice. Neutralization of TNF reduced apoptosis of leukocytes and splenocytes, increased production of IFN-gamma by NK cells, increased phagocytosis in the spleen, decreased spirochetemia, and rescued mice from early death. Our results indicate that at times of high pathogen load, as during peak bacteremia in relapsing fever borreliosis, IL-10 protects innate immune cells from apoptosis via inhibition of TNF resulting in improved pathogen control.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

Innate immunity mediates follicular transport of particulate but not soluble protein antigen

Ag retention on follicular dendritic cells (FDCs) is essential for B cell activation and clonal selection within germinal centers. Protein Ag is deposited on FDCs after formation of immune complexes with specific Abs. In this study, by comparing the same antigenic determinant either as soluble protein or virus-like particle (VLP), we demonstrate that VLPs are transported efficiently to murine splenic FDCs in vivo in the absence of prior immunity. Natural IgM Abs and complement were required and sufficient to mediate capture and transport of VLPs by noncognate B cells. In contrast, soluble protein was only deposited on FDCs in the presence of specifically induced IgM or IgG Abs. Unexpectedly, IgG Abs had the opposite effect on viral particles and inhibited FDC deposition. These findings identify size and repetitive structure as critical factors for efficient Ag presentation to B cells and highlight important differences between soluble proteins and viral particles.     

Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used alpha1,3-galactosyltransferase-deficient (GalT(-/-)) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galalpha1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT(-/-) and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT(-/-) and (for non-Gal specificities) wild-type mice. These cells are Mac-1(-) but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT(-/-) mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT(-/-), Ig micro -chain mutant ( micro (-/-)) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT(-/-) mice. When sorted GalT(-/-) Mac-1(+) peritoneal cavity B cells were adoptively transferred to B6 GalT(-/-), micro (-/-) mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1(-) B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1(+) B-1 cells are precursors of Mac-1(-) splenic IgM Ab-secreting cells.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Persistent brain infection and disease reactivation in relapsing fever borreliosis

Relapsing fever, an infection caused by Borrelia spirochetes, is generally considered a transient, self-limiting disease in humans. The present study reveals that murine infection by Borrelia duttonii can be reactivated after an extended time as a silent infection in the brain, with no bacteria appearing in the blood and spirochete load comparable to the numbers in an infected tick. The host cerebral gene expression pattern is indistinguishable from that of uninfected animals, indicating that persistent bacteria are not recognized by the immune system nor cause noticeable tissue damage. Silent infection can be reactivated by immunosuppression, inducing spirochetemia comparable to that of initial densities. B. duttonii has never been found in any host except man and the tick vector. We therefore propose the brain to be a possible natural reservoir of the spirochete. The view of relapsing fever as an acute disease should be extended to include in some cases prolonged persistence, a feature characteristic of the related spirochetal infections Lyme disease and syphilis.     

Macrophages,but not T and B lymphocytes,are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation

Bullous pemphigoid (BP) is a subepidermal blistering disease associated with autoantibodies against two hemidesmosomal proteins, BP180 and BP230. Numerous inflammatory cells infiltrate the upper dermis in BP. We have previously shown by passive transfer studies that Abs to the ectodomain of murine BP180 are capable of triggering blisters in mice that closely mimic human BP. Experimental BP depends on complement activation and neutrophil infiltration. In the present study, we investigated the relative contribution of neutrophils, mast cells (MCs), macrophages (Mphi), and lymphocytes and their functional relationship in the immunopathogenesis of this disease model by using mice deficient in these cells. Wild-type, T cell-deficient, and T and B cell-deficient mice injected intradermally with pathogenic anti-murine BP180 IgG exhibited extensive subepidermal blisters. In contrast, mice deficient in neutrophils, MCs, and Mphi were resistant to experimental BP. MCs play a major role in neutrophil recruitment into the dermis. Furthermore, Mphi-mediated neutrophil infiltration depends on MC activation/degranulation.     

The resolution of relapsing fever borreliosis requires IgM and is concurrent with expansion of B1b lymphocytes

The rate of pathogen clearance is a critical determinant of morbidity and mortality. We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii. SCID or Rag(-/-) mice were incapable of resolving B. hermsii infection, indicating a critical role for T and/or B cells. TCR(-/-) mice, which lack T cells, and IL-7(-/-) mice, which are deficient in both T cells and follicular B cells, but not in B1 cells and splenic marginal zone (MZ) B cells, efficiently cleared B. hermsii. These findings suggested that B1 cells and/or MZ B cells, two B cell subsets that are known to participate in rapid, T-independent responses, might be involved. The efficient resolution of the episodes of moderate level bacteremia by splenectomized mice suggested that MZ B cells do not play the primary role in clearance of this bacterium. In contrast, xid mice, which are deficient in B1 cells, suffered more severe episodes of bacteremia than wild-type mice. The hypothesis that B1 cells are critical for clearance of B. hermsii was further supported by a selective expansion of the B1b (i.e., IgM(high), IgD(-/low), Mac1(+) CD23(-), and CD5(-)) cell subset in infected xid mice, which coincided with the eventual resolution of infection. Finally, mice selectively incapable of secreting IgM, the dominant isotype produced by B1 cells, were completely unable to clear B. hermsii. Together these results support the model that B1b cells generate the T-independent IgM required for the control and resolution of relapsing fever borreliosis.     

Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice

Group B Streptococcus (GBS) is the foremost bacterial cause of serious neonatal infections. Protective immunity to GBS is mediated by specific Abs to the organism's capsular polysaccharide Ags. To examine the role of complement in the humoral immune response to type III GBS capsular polysaccharide (III-PS), mice deficient in C3 or in CD21/CD35 (i.e., complement receptors 1 and 2; CR1/CR2) were immunized with III-PS. Mice deficient in C3 or Cr2 had an impaired primary immune response to III-PS. The defective response was characterized by low IgM levels and the lack of an isotype switch from IgM to IgG Ab production. Compared with wild-type mice, C3- and Cr2-deficient mice exhibited decreased uptake of III-PS by follicular dendritic cells within the germinal centers and impaired localization of III-PS to the marginal zone B cells. Complement-dependent uptake of capsular polysaccharide by marginal zone B cells appears necessary for an effective immune response to III-PS. The normal immune response in wild-type mice may require localization of polysaccharide to marginal zone B cells with subsequent transfer of the Ag to follicular dendritic cells.     

Ig knock-in mice producing anti-carbohydrate antibodies: breakthrough of B cells producing low affinity anti-self antibodies

Natural Abs specific for the carbohydrate Ag Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) play an important role in providing protective host immunity to various pathogens; yet little is known about how production of these or other anti-carbohydrate natural Abs is regulated. In this study, we describe the generation of Ig knock-in mice carrying functionally rearranged H chain and L chain variable region genes isolated from a B cell hybridoma producing alphaGal-specific IgM Ab that make it possible to examine the development of B cells producing anti-carbohydrate natural Abs in the presence or absence of alphaGal as a self-Ag. Knock-in mice on a alphaGal-deficient background spontaneously developed alphaGal-specific IgM Abs of a sufficiently high titer to mediate rejection of alphaGal expressing cardiac transplants. In the spleen of these mice, B cells expressing alphaGal-specific IgM are located in the marginal zone. In knock-in mice that express alphaGal, B cells expressing the knocked in BCR undergo negative selection via receptor editing. Interestingly, production of low affinity alphaGal-specific Ab was observed in mice that express alphaGal that carry two copies of the knocked in H chain. We suggest that in these mice, receptor editing functioned to lower the affinity for self-Ag below a threshold that would result in overt pathology, while allowing development of low affinity anti-self Abs.     

Invariant NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1 cells to initiate contact sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.     

Pathogenic natural antibodies propagate cerebral injury following ischemic stroke in mice

Self-reactive natural Abs initiate injury following ischemia and reperfusion of certain tissues, but their role in ischemic stroke is unknown. We investigated neoepitope expression in the postischemic brain and the role of natural Abs in recognizing these epitopes and mediating complement-dependent injury. A novel IgM mAb recognizing a subset of phospholipids (C2) and a previously characterized anti-annexin IV mAb (B4) were used to reconstitute and characterize injury in Ab-deficient Rag1(-/-) mice after 60 min of middle cerebral artery occlusion and reperfusion. Reconstitution with C2 or B4 mAb in otherwise protected Rag1(-/-) mice restored injury to that seen in wild-type (wt) mice, as demonstrated by infarct volume, demyelination, and neurologic scoring. IgM deposition was demonstrated in both wt mice and reconstituted Rag1(-/-) mice, and IgM colocalized with the complement activation fragment C3d following B4 mAb reconstitution. Further, recombinant annexin IV significantly reduced infarct volumes in wt mice and in Rag1(-/-) mice administered normal mouse serum, demonstrating that a single Ab reactivity is sufficient to develop cerebral ischemia reperfusion injury in the context of an entire natural Ab repertoire. Finally, C2 and B4 mAbs bound to hypoxic, but not normoxic, human endothelial cells in vitro. Thus, the binding of pathogenic natural IgM to postischemic neoepitopes initiates complement-dependent injury following murine cerebral ischemia and reperfusion, and, based also on previous data investigating IgM reactivity in human serum, there appears to be a similar recognition system in both mouse and man.     

Cellular immune responses are essential for the development of Helicobacter felis-associated gastric pathology.

The bacteria Helicobacter pylori is a major human pathogen that infects over half of the world's population. Infection initiates a series of changes in the gastric mucosa, beginning with atrophic gastritis and leading in some patients to peptic ulcer disease, mucosa-associated lymphomas, and gastric adenocarcinoma. Although this cascade of events clearly occurs, little is known about the role of the host immune response in disease progression. We have utilized the C57BL/6 Helicobacter felis mouse model to critically analyze the role of the adaptive immune response in the development of Helicobacter-associated gastric pathology. Infection of B and T cell-deficient RAG-1-/- mice or T cell-deficient TCRbetadelta-/- mice with H. felis resulted in high levels of colonization, but no detectable gastric pathology. Conversely, infection of B cell-deficient microMT mice resulted in severe gastric alterations identical with those seen in immunocompetent C57BL/6-infected mice, including gastric mucosal hyperplasia and intestinal metaplasia. These results demonstrate that the host T cell response is a critical mediator of Helicobacter-associated gastric pathology, and that B cells and their secreted Abs are not the effectors of the immune-mediated gastric pathology seen after H. felis infection. These results indicate that in addition to specific Helicobacter virulence factors, the host immune response is an important determinant of Helicobacter-associated disease.     

Immunoprevention of mammary carcinoma in HER-2/neu transgenic mice is IFN-gamma and B cell dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185(neu) completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185(neu) Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-gamma production (IFN-gamma(-/-)) or with B cell-deficient mice (microMT). Vaccination did not protect NeuT-IFN-gamma(-/-) mice, thus confirming a central role of IFN-gamma. The block of Ab production in NeuT-microMT mice was incomplete. About one third of NeuT-microMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-microMT mice that responded to the vaccine with a robust production of anti-p185(neu) Ab displayed a markedly delayed tumor onset. In these NeuT-microMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-microMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2(q) molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.     

Epidemiology of relapsing fever borreliosis in Europe

Tick-borne relapsing fever is a bacterial infection caused by spirochetes of the genus Borrelia. This zoonotic disease is transmitted to humans through the bite of soft ticks of the genus Ornithodoros. It is responsible for recurring fever access associated with spirochetemia. We present here an overview of tick-borne relapsing fever occurring in Europe, as well as of the potential threat to travellers.     

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

Critical role for IgM in host protection in experimental filarial infection

We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM-/-), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM-/- mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.     

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.