Ivana Kawikova

Early Local Generation of C5a Initiates the Elicitation of Contact Sensitivity by Leading to Early T Cell Recruitment

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR−/−) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-γ mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR−/− macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1α and -1β, IFN-γ-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR−/− mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.

B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.

Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell–derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.

Increased serum levels of interleukin-12 and tumor necrosis factor-alpha in Tourette’s syndrome

BACKGROUND The hypothesis that common infections can modulate the onset and course of tic disorders and early-onset obsessive-compulsive disorder (OCD) in pediatric populations is longstanding. To date, most investigations have focused on the hypothesis of molecular mimicry and humoral immune responses. This study was carried out to investigate whether cytokines associated with the innate immune response or T cell activation were altered under baseline conditions and during periods of symptom exacerbation. METHODS Forty-six patients with Tourettes syndrome and/or early-onset OCD, aged 7-17 years, and 31 age-matched control subjects participated in a prospective longitudinal study. Ratings of clinical severity and serum were collected at regular intervals, and serum concentrations of 10 cytokines were measured repeatedly. RESULTS Interleukin-12 and tumor necrosis factor alpha concentrations at baseline were elevated in patients compared with control subjects. Both of these markers were further increased during periods of symptom exacerbation. CONCLUSIONS These findings suggest that symptom exacerbations are associated with an inflammatory process propagated by systemic and local cytokine synthesis that might involve the central nervous system. We conclude that, in the future, longitudinal studies of children with neuropsychiatric disorders should examine the involvement of innate and T cell immunity.

Streptococcal upper respiratory tract infections and exacerbations of tic and obsessive-compulsive symptoms: A prospective longitudinal study

OBJECTIVE The objective of this blinded, prospective, longitudinal study was to determine whether new group A β hemolytic streptococcal (GABHS) infections are temporally associated with exacerbations of tic or obsessive-compulsive (OC) symptoms in children who met published criteria for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). A group of children with Tourette syndrome and/or OC disorder without a PANDAS history served as the comparison (non-PANDAS) group. METHOD Consecutive clinical ratings of tic and OC symptom severity were obtained for 31 PANDAS subjects and 53 non-PANDAS subjects. Clinical symptoms and laboratory values (throat cultures and streptococcal antibody titers) were evaluated at regular intervals during a 25-month period. Additional testing occurred at the time of any tic or OC symptom exacerbation. New GABHS infections were established by throat swab cultures and/or recent significant rise in streptococcal antibodies. Laboratory personnel were blinded to case or control status, clinical (exacerbation or not) condition, and clinical evaluators were blinded to the laboratory results. RESULTS No group differences were observed in the number of clinical exacerbations or the number of newly diagnosed GABHS infections. On only six occasions of a total of 51 (12%), a newly diagnosed GABHS infection was followed, within 2 months, by an exacerbation of tic and/or OC symptoms. In every instance, this association occurred in the non-PANDAS group. CONCLUSIONS This study provides no evidence for a temporal association between GABHS infections and tic/OC symptom exacerbations in children who meet the published PANDAS diagnostic criteria.

Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/γc−/−, Balb/c-Rag1−/−γc−/−, and C.B-17-scid/bg immunodeficient mice

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

Decreased numbers of regulatory T cells suggest impaired immune tolerance in children with tourette syndrome : A preliminary study

BACKGROUND Post-streptococcal autoimmune inflammation of basal ganglia was suggested to be an etiological factor in some cases of Tourette syndrome (TS). Since regulatory T (T reg) cells play a major role in preventing autoimmunity, we hypothesized that a defect in T reg cells may be present in children with TS. We also postulated that group A beta hemolytic streptococcal infections could promote autoimmune responses by releasing exotoxins (streptococcal pyrogenic exotoxins [SPE]). METHODS We analyzed peripheral blood of TS patients and healthy age-matched control subjects by fluorescence-activated cell sorting (FACS) on multiple occasions and determined the numbers of CD4(+)CD25(+)CD69(-) T reg cells. Further, we quantified the number of CD4(+) and CD8(+) lymphocytes with regard to Vbeta chains to which SPEs are known to bind. RESULTS A significant decrease in T reg cells was observed in patients with moderate to severe TS symptoms compared with healthy age-matched control children. A decrease in T reg cell number was also noted during symptom exacerbations in five out of six patients. Further, we found a significant decrease in numbers of CD8(+)Vbeta18(+) T cells in moderate to severe TS patients. CONCLUSIONS These data support our hypothesis that at least some TS patients may have a decreased capacity to inhibit autoreactive lymphocytes through a deficit in T reg cells. Interactions of host T cell immunity and microbial factors may also contribute to the pathogenesis of TS.

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 μMT 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.

Elevated expression of MCP-1, IL-2 and PTPR-N in basal ganglia of Tourette syndrome cases

BACKGROUND Post-infectious autoimmunity has been implicated in pathogenesis of Tourettes syndrome (TS) but no evidence of inflammation in central nervous system has been reported yet. We evaluated the expression of genes encoding selected inflammatory factors in post-mortem specimen of adult TS patients: interferon-γ (a cytokine released from CD8 and Thelper 1 CD4 subset of T lymphocytes), interleukin-2 (IL-2, a growth factor derived from T lymphocytes), interleukin-1 β (a cytokine involved in initiation of inflammation), monocyte chemotactic factor -1 (MCP-1, a marker of chronic inflammation) and CD45 (pan-leukocytic marker). For validation purposes, we determined expression of three genes that were previously reported to be elevated in post-mortem specimen of other TS cases: protein tyrosine phosphatase receptor-N (PTPR-N), PTPR-U and recoverin. METHODS Total RNA was isolated from formalin fixed brain tissue sections of basal ganglia area from four patients with TS and four control subjects, and real-time reverse transcription-polymerase chain reaction analysis was employed to quantitatively evaluate gene expression of the selected genes. RESULTS Significantly increased expression of MCP-1, IL-2 and PTPR-N was observed in TS cases (6.5-fold, 2.3-fold and 16.1-fold increase, respectively, p<0.05). CONCLUSIONS Elevated expression of MCP-1 and IL-2 supports the possibility of chronic inflammatory processes in the basal ganglia. Replication of elevated expression of PTPR-N in TS specimen suggests that pathway(s) involving this molecule may be important in TS pathogenesis.

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-γ via CXCR3 chemokines

We investigated the role of T helper (Th)1‐ and Th2‐type cytokines in delayed‐type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freunds adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen‐specific eosinophil‐rich responses were elicited in wild‐type mice, but not in T‐cell receptor‐α–/– mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)‐4, IL‐5 and interferon‐γ (IFN‐γ). IFN‐γ‐dependent specific immunoglobulin G (IgG)2a and IL‐4‐dependent IgG1 were also generated. Delayed‐type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL‐5–/–, IL‐4–/– and signal transducer and activator of transcription‐6 (STAT‐6)–/– mice, and with anti‐IL‐4 treatment of wild‐type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN‐γ–/– mice, and IFN‐γ protein and the IFN‐γ‐inducible CXC chemokine, IP‐10, were present in 24‐hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP‐10. These results suggest that both a Th2‐like (IL‐5, IL‐4 and STAT‐6) and a Th1‐like (IFN‐γ, IP‐10, CXCR3) pathway contribute to eosinophil recruitment in early delayed‐type hypersensitivity.

Children with Tourette's syndrome may suffer immunoglobulin A dysgammaglobulinemia: preliminary report.

BACKGROUND Postinfectious autoimmunity has been implicated in Tourettes syndrome and obsessive-compulsive disorder (TS/OCD), whereas increased frequency of upper respiratory tract infections (URTI) in TS/OCD patients suggests immune deficiency. We hypothesized that antineuronal antibodies may be elevated in patients (reflecting autoimmune processes), and levels of total immunoglobulins (Igs) may be decreased (reflecting immune deficiency). METHODS We analyzed plasma of TS/OCD patients (n = 24) and healthy age- and sex-matched control subjects (n = 22) by enzyme-linked immunosorbent assay (ELISA) for the levels of total and specific IgG, IgM, and IgA against antigens previously identified in multiple sclerosis (myelin basic protein and myelin-associated glycoprotein) and Sydenhams chorea (ganglioside-GM1, lysoganglioside, and tubulin). RESULTS Total IgA was decreased in TS/OCD patients (median 115 mg/100 mL) compared with control subjects (141 mg/100 mL; p = .02). Specific IgA against all antigens, except tubulin were also decreased in the patients (MPB 0 vs. 13 [ELISA units [EU]; myelin-associated glycoprotein 29 vs. 44 EU, p = .04; ganglioside GM1 21 vs. 35 EU, p = .01; lysoganglioside 44 vs. 56 EU, p = .03; tubulin 44 vs. 44 EU, p = .8). The levels of total IgA and anti-myelin basic protein (MBP) IgA were significantly lower in the subgroup of pediatric autoimmune neuropsychiatric disorder associated with Streptococcus (PANDAS) cases (n = 10) than in non-PANDAS cases (n = 9; total IgA 98 mg/100 mL vs. 133 mg/mL, p = .03; anti-MBP IgA 1 vs. 6 EU, p = .03) or healthy control subjects (total IgA 141 mg/100 mL, p = .02; anti-MBP IgA 13 EU, p = .005). CONCLUSIONS At least some TS/OCD patients may suffer IgA dysgammaglobulinemia, possibly rendering the children more prone to URTI.