Ultrastructure of mouse peritoneal cells engaged in spontaneous secretion of antibody against mouse and sheep erythrocytes and evolution of cell types during culture.

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG)² in incomplete Freund's adjuvant (IFA) reduced both the incidence and severity of experimental autoimmune thyroiditis (EAT) in animals subsequently challenged with GPTG in complete Freund's adjuvant (CFA). Antithyroglobulin antibody titers were reduced by pretreatment with GPTG in IFA in some, but not all, experiments while delayed hypersensitivity to GPTG was not affected. The suppressive effect induced by antigen pretreatment was transferrable by lymphoid cells but not by serum from pretreated animals.     

Suppression of experimental autoimmune thyroiditis in guinea pigs by pretreatment with thyroglobulin-coupled spleen cells

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG) coupled to syngeneic spleen cells (GPTG-SC) suppressed the development of experimental autoimmune thyroiditis (EAT) induced by immunization with GPTG in complete Freund's adjuvant (CFA). Antibody titers to GPTG were only minimally suppressed in GPTG-SC pretreated animals. GPTG-SC also suppressed the sensitization of periotneal exudate T lymphocytes which proliferate in vitro in the presence of GPTG.     

In vivo cyclophosphamide-mediated augmentation and aqueous HGG-induced suppression of murine delayed hypersensitivity to HGG are reflected by in vitro HGG-stimulated proliferation of lymph node cells from equivalent mice

The question addressed in this report was whether immunological processes which culminate in in vivo expression and augmentation as well as suppression of delayed effector cell activity are mirrored by events that can be quantified in vitro. For this purpose the previously characterized murine model of delayed hypersensitivity (DHS), which employs SJL/J mice immunized with aggregated human γ-globulin (AHGG) in complete Freund's adjuvant (CFA) was employed. The results indicated that lymph node cells (LNC) from cyclophosphamide (CY)-pretreated, AHGG-CFA immunized mice expressed increased proliferation in the presence of HGG and concanavalin A (Con A) but decreased LPS responsiveness compared with LNC from equivalently immunized but non-CY-treated animals. It was also found that LNC from CY-treated, AHGG-CFA immunized mice that were pretreated with aqueous HGG (aqHGG), but not aqueous bovine serum albumin (aqBSA), evidenced a markedly decreased capacity to proliferate in the presence of HGG compared with LNC from equivalent animals that were not pretreated with aqHGG. This suppressive effect was not attributable to antibody production. These findings support the conclusion that in vitro quantitation of antigen-induced proliferation by LNC from HGG-DHS mice appears to correlate with modulatory effects which are observed in in vivo expression of DHS responses.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Chronic relapsing experimental allergic encephalomyelitis: Identification and dynamics of T and B cells within the central nervous system

Using a monoclonal antibody against guinea pig T cells and anti-guinea pig immunoglobulins, T- and B-cell dynamics were studied by immunofluorescence in situ in the central nervous system (CNS) of animals with untreated and treated chronic relapsing experimental allergic encephalomyelitis (EAE). Treated animals were given a series of injections of either myelin basic protein (MBP) in incomplete Freund's adjuvant (IFA) or MBP and galactocerebroside in IFA. Within the CNS, T and B cells showed distinct distribution patterns in untreated chronic relapsing EAE, similar to that recently described in acute EAE. T cells were predominantly localized within the CNS parenchyma and B cells were mainly found in perivascular areas. B-cell infiltrates were more extensive than in acute EAE and, although most were centered around blood vessels, some were also detectable in the parenchyma. IgG, C₃, and albumin deposits were common. These observations suggest an age-dependent difference in the immune response. In treated chronic EAE, the disease process was apparently arrested and T- and B-cell infiltrates in the white matter were negligible. Therefore, it appears that the present treatment protocol prevents lymphocytes from entering the CNS parenchyma.     

Rat leukocyte inhibitory factor (LIF): Similarities to human LIF and generation by cells from rats with collagen-induced arthritis

Rat lymph node cells (LNC) produce a mediator which exhibits functional and physicochemical similarities to human leukocyte inhibitory factor (LIF). Measurement of LIF can be used to quantify cellular sensitivity in rats to foreign protein antigens or to a heterologous antigen in vitro. Concanavalin A-induced rat LIF has a molecular weight of 100,000-60,000 daltons and retains activity after heating to 56 °C for 30 min. Rat LIF is not synthesized in the presence of puromycin and appears to be a protein, since it is inactivated by treatment with chymotrypsin. Moreover, the activity of rat LIF is susceptible to the serine esterase inhibitor, diisopropylphosphofluoridate, but it is resistant to neuraminidase treatment. Finally, rat LIF preferentially inhibits the migration of rat and human polymorphonuclear leukocytes but not that of rat or guinea pig peritoneal exudate cells enriched for macrophages. Except for the more dispersed size of rat LIF, these properties are analogous to those described for human LIF. LIF activity is generated, in an antigen-specific manner, by LNC sensitized to ovalbumin or purified protein derivative of tuberculin when cultured with the antigen used for sensitization. LNC from rats rendered arthritic by prior intradermal (id) injection of native chick type II collagen in incomplete Freund's adjuvant produce LIF in response to this heterologous antigen. These studies delineate a new assay for cellular sensitivity in rats and provide additional evidence that cellular reactivity to type II collagen is present in this animal model of arthritis.     

Pineal function in hamsters bearing melatonin antibodies

Adult male hamsters were immunized with a melatonin-thyroglobulin conjugate emulsified in Freund's adjuvant; control animals were injected with Freund's adjuvant only or were untreated. Blinding of control animals resulted in marked testicular atrophy. The testes of sighted, antibody-bearing hamsters exhibited testicular atrophy and blinding appeared to exacerbate the degree of involution. These results suggest that the pineal gland may secrete a substance which, under conditions of diminished or absent circulating levels of biologically active melatonin, is antigonadotropic and whose secretion may be enhanced by dark-induced stimulation of the pineal gland.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

Experimental uveitis induced by products of activated lymphocytes: Intraocular effects of rhodopsin-induced lymphokines

Immunization of strain 13 guinea pigs by footpad injection of bovine rhodopsin in complete Freund's adjuvant produces experimental autoimmune uveitis (retinopathy) with retinal pathology characterized by destruction of the retinal photoreceptor cell layer, as we reported previously. Since rhodopsin-induced EAU can be passively transferred by immune cells but not antirhodopsin antibody, the present studies were conducted to determine if soluble products of activated lymphocytes (PAL) could cause the retinal pathology seen in experimental uveitis. These studies, reported here, show that intravitreal injection of supernatant factors generated by guinea pig lymph node cells specifically activated in vitro with rhodopsin or purified protein derivatives of tuberculin or nonspecifically activated with the mitogen concanavalin A produce uveitis in the normal guinea pig eye. The PAL produced mononuclear cell infiltration in the vitreous and a retinopathy with loss of retinal photoreceptor cells. Inflammatory cell infiltrates were found in the retina but not found in the anterior segment of the eye. Control lymphocyte supernatants did not produce retinal pathology. In guinea pigs sensitized to rhodopsin-complete Freund's adjuvant, intravitreal injection of the PAL produced a mononuclear vitreal infiltrate, disruption of the photoreceptor cell layer, necrosis of the inner retina, and a granulomatous infiltrate in the choroid with damage to the retinal pigment epithelium. Injection of control supernatants into the rhodopsin-complete Freund's adjuvant sensitized guinea pig eye did not produce inflammation and the retinal photoreceptor cell layer remained intact. Results from our studies indicate a role for the PAL along with other, yet undefined, factors in the initiation of autoimmune uveitis and in the autoimmune pathology of the retina.     

In vitro response to basic protein in experimental allergic encephalomyelitis: effect of pretreatment with basic protein in incomplete adjuvant

Guinea pigs injected with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) fail to develop experimental allergic encephalomyelitis (EAE) after challenge with BP in complete Freund's adjuvant (CFA). Such protected animals fail to manifest significant in vitro lymphocyte proliferative responses to BP 13 days after BP/CFA in comparison to animals with EAE. Other BP/IFA-BP/CFA animals develop significant albeit modest responses to BP 21 and 28 days after BP/CFA but do not develop EAE. There was little effect on the response to tuberculin (OT). Guinea pigs receiving only BP/IFA develop a modest transient reactivity to BP and no EAE. CFA alone after BP/IFA elicits a normal response to OT and has no effect on the response to BP.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

The induction of cell-mediated immunity and tolerance to insulin with insulin coupled to syngeneic lymphoid cells

The induction of delayed type hypersensitivity (DTH) and tolerance to DTH against bovine insulin in mice were explored. DTH was induced with insulin in complete Freund's adjuvant (CFA) and was assessed by ear swelling in vivo and by antigen-driven cell proliferation in vitro. Using the concept that thymus cell unresponsiveness is most easily accomplished via antigen on syngeneic membranes, tolerance was induced by iv injection of syngeneic lymphoid cells which had been coupled to insulin with carbodiimide. Mice tolerized with insulin-coupled cells and then sensitized with insulin-CFA had diminished ear swelling in vivo and decreased insulin-driven cell proliferation in vitro. This unresponsiveness was antigen specific but was also inconstant in degree with regard to suppression of ear swelling, most likely because of variability in coupling of insulin to cells. Proliferative responses were more uniformly suppressed, suggesting the possibility that two target cells were being tolerized. Thus, as with other proteins, the biologically active insulin can be used to induce tolerance.     

Ascaris suum and Toxocara canis: Egg extract antigens in guinea pigs and the macrophage migration inhibition test

Guinea pigs immunized with 500 μg of either Ascaris suum or Toxocara canis egg extracts, emulsified with Freund's complete adjuvant, were skin tested with both the homologous and heterologous antigens. Cross-reactions were observed in both groups. Migration of macrophages from sensitized animals was more inhibited by homologous than by heterologous antigens. Lymph-node lymphocytes from sensitized animals were stimulated to incorporate [⁸H]thymidine similarly by both antigens.     

Effect in neonatal thymectomy on experimental autoimmune thyroiditis in guinea pigs.

These studies examined the effect of neonatal thymectomy on the induction of experimental autoimmune thyroiditis (EAT) in the guinea pigs. Thymectomy was found to result in a consistent and profound inhibition of the development of lesions of EAT in both strain 2 and strain 13 guinea pigs. Thymectomized guinea pigs also had reduced antibody titers to guinea pig thyroglobulin (GPTG), while delayed hypersensitivity reactions to GPTG were less markedly affected by thymectomy. Thymectomized guinea pigs had significant functional peripheral T cells as evidenced by normal responses of lymph node cells to T cell mitogens. These results indicate that a T cell subpopulation which is sensitive to neonatal thymectomy is required for the development of EAT and antithyroglobulin antibody in the guinea pig.     

The suppression of experimental allergic encephalomyelitis in Lewis rats by treatment with myelin basic protein-cell conjugates

Pretreatment of Lewis rats with guinea pig (GP) myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) suppressed the subsequent induction of experimental allegic encephalomyelitis (EAE) with GP-MBP in Freund's complete adjuvant. The degree of suppression correlated positively with the amount of antigen coupled to the SL. GP-MBP coupled to syngeneic red blood cells (RBC) also resulted in suppression of EAE and the extent of the suppression was related to the dose of cells. These regimens of pretreatment also resulted in a decrease in the in vitro lymphocyte proliferative response to GP-MBP and in the extent of perivascular cuffing in the spinal cord. No decrease in the anti-MBP antibody response was detected in rats pretreated with either GP-MBP-SL or GP-MBP-RBC conjugates. Transfer of lymph node cells from rats pretreated with GP-MBP-RBC resulted in a decrease in disease severity in recipients. It is concluded that prior administration of MBP-cell conjugates is an effective way of suppressing the symptoms of EAE.     

Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis

Introduction

Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model.

Methods

DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression.

Results

Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA/CII group and IL-1β, tumor necrosis factor-α, transforming growth factor-β, IL-6 and IL-23 in the IFA/CII group.

Conclusions

Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.     

Enhanced iodination of thyroglobulin facilitates processing and presentation of a cryptic pathogenic peptide

Increased iodine intake has been associated with the development of experimental autoimmune thyroiditis (EAT), but the biological basis for this association remains poorly understood. One hypothesis has been that enhanced incorporation of iodine in thyroglobulin (Tg) promotes the generation of pathogenic T cell determinants. In this study we sought to test this by using the pathogenic nondominant A(s)-binding Tg peptides p2495 and p2694 as model Ags. SJL mice challenged with highly iodinated Tg (I-Tg) developed EAT of higher severity than Tg-primed controls, and lymph node cells (LNC) from I-Tg-primed hosts showed a higher proliferation in response to I-Tg in vitro than Tg-primed LNC reacting to Tg. Interestingly, I-Tg-primed LNC proliferated strongly in vitro against p2495, but not p2694, indicating efficient and selective priming with p2495 following processing of I-Tg in vivo. Tg-primed LNC did not respond to either peptide. Similarly, the p2495-specific, IL-2-secreting T cell hybridoma clone 5E8 was activated when I-Tg-pulsed, but not Tg-pulsed, splenocytes were used as APC, whereas the p2694-specific T cell hybridoma clone 6E10 remained unresponsive to splenic APC pulsed with Tg or I-Tg. The selective in vitro generation of p2495 was observed in macrophages or dendritic cells, but not in B cells, suggesting differential processing of I-Tg among various APC. These data demonstrate that enhanced iodination of Tg facilitates the selective processing and presentation of a cryptic pathogenic peptide in vivo or in vitro and suggest a mechanism that can at least in part account for the association of high iodine intake and the development of EAT.     

The Extracellular Domain of Myelin Oligodendrocyte Glycoprotein Elicits Atypical Experimental Autoimmune Encephalomyelitis in Rat and Macaque Species

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.     

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin.

The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund's adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.