Kenneth W. Beagley

Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice.

BACKGROUND/AIMS Oral administration of dextran sulfate sodium (DSS) has been reported to induce colitis in mice. The purpose of this study was to determine whether the possible pathogenic mechanism involved the acquired immune system. METHODS Normal BALB/c and related C.B17 severe combined immunodeficient mice were fed 5% DSS (40 kilodaltons) in their drinking water for 7 days; controls were fed only water. Colons were scored for histological activity at various times. Cytokine production by cultures of colon and of draining lymph node cell was measured. The effect of DSS on the proliferation of the MCA-38 colonic epithelial cell line was assessed. RESULTS DSS feeding resulted in a very reproducible acute distal colitis in both BALB/c and C.B17 severe combined immunodeficient mice. The lesions of BALB/c mice had an increased production of macrophage-derived cytokines, such as interleukin (IL) 1 beta, IL-6, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor, but not the T-cell cytokines IL-3 or interferon gamma. Draining lymph node cells produced these cytokines plus interferon gamma and IL-3. DSS inhibited MCA-38 cells at doses that would be easily achieved in the distal colon. CONCLUSIONS Acute DSS-induced colitis does not require the presence of T cells or B cells because it occurred in C.B17 severe combined immunodeficient mice that lack these cells. Its induction may result from a toxicity of DSS for colonic epithelial cells.

Detection of individual mouse splenic T cells producing IFN-γ and IL-5 using the enzyme-linked immunospot (ELISPOT) assay

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.

Peyer's patch B cells with memory cell characteristics undergo terminal differentiation within 24 hours in response to interleukin-6

Culture of Peyers patch (PP) B cells with interleukin-6 (IL-6) for 7 days results in a six- to eightfold increase in secretion of IgA, while little or no increase in IgM or IgG secretion occurs in these cultures. Further, greater than 80% of IgA is produced within the first 72 h of culture. Using a sensitive enzyme-linked immunospot (ELISPOT) assay, we have shown that culture of PP B cells with IL-6 for 24 h gave increased IgA spot-forming cells (SFC) (4- to 6- fold) even though secreted IgA, as measured by RIA, had only increased 1.6- to 2.0-fold. In addition, significant increases in IgA SFC numbers could be demonstrated as early as 4 h after addition of IL-6. The increase in IgA secretion was not the result of IL-6-induced B-cell proliferation, since culture of B cells with IL-6 resulted in no increase in [3H]thymidine incorporation compared to untreated controls. This was supported by studies with mitomycin C which, when added to B cell cultures, had no effect on the IL-6-induced increase in numbers of IgA SFC. Increased IgA secretion was totally abolished by actinomycin D, an inhibitor of RNA transcription, showing that continued production of alpha mRNA is essential for IL-6-induced IgA secretion. Separation of PP B cells into peanut agglutin (PNA)Hi (germinal center [GC]) and PNALo (non-GC) subpopulations before culture with IL-6 showed that only PNALo B cells transcribe increased levels of alpha mRNA message and secrete high levels of IgA in response to this cytokine. Although the GC are the site of B-cell proliferation and presumably of switching to IgA and contain 70 to 85% of sIgA+ B cells in the PP, these PNAHi B cells do not respond to IL-6. This suggests that memory sIgA+ B cells in PP express IL-6 receptor (IL-6R) and respond to this cytokine with rapid differentiation into plasma cells that secrete IgA.

Role of Interleukin-6 in Human and Mouse Mucosal IgA Plasma Cell Responses

SummaryIn summary, we have shown that human appendix and murine PP B cells, freshly isolated from normal tissue, respond to IL-6 with significant increases in IgA SFC. Further, sIGA+ B cells from appendix express more IL-6R than is seen with B cells isolated from PBMC and spleen. When IgA subclass responses were measured, rhIL-6 induced both IgA1 and IgA2 SFC responses; however, 60–70% of the total response was represented by the IgA2 subclass. Our studies suggest that the human appendix as well as murine PP are enriched sources for sIgA+ B cells which are responsive to cytokines such as IL-6 for induction of IgA plasma cell responses.

Substance P Promotes Peyer’s Patch and Splenic B Cell Differentiation

Current interpretations of immune processes tends to exclude nonhematopoietic cells and their representative mediators as possible contributors to the immune response. One such excluded element is the nervous system. To date, there is considerable evidence that the sympathetic1 and the peptidergic2 nervous systems substantiate neural involvement in immune regulation. This is never more evident than the effects exerted by neuropeptides upon mucosal immune responses. One such neuropeptide is substance P (SP). A member of the tachykinin family, SP is an 11 amino acid peptide3, and outside the brain, it is found in greatest concentrations in the gut.3 SP is a product of sensory ganglion cells, and it is transported to peripheral sites where it is stored and released upon noxious stimulation.3 SP-containing fibers have been localized in the Peyer’s patches (PP)4 suggesting possible influences upon B cell differentiation.

Role of IL-6 in human antigen-specific and polyclonal IgA responses.

It is now well established that several cytokines are involved in B cell activation, proliferation, and differentiation. From earlier studies, it became clear that some of these cytokines acted later in B cell responses and contributed to terminal differentiation and immunoglobulin synthesise1. A prominent cytokine among these was a factor originally termed B cell-differentiation factor, which could be distinguished from B cell-stimulating factor-1 (BSF-1), or IL-4, and was subsequently termed BSF-22,3. When BSF-2 was purified and subsequently cloned3–5, it was shown to be identical to other cloned proteins, e.g., interferon beta (IFN-β)6, the 26kDa protein7, and hybridoma growth factor8–10, and was renamed IL-6.

FACS-Sorted Spleen and Peyer’s Patch Dendritic Cells Induce Different Responses in Th0 Clones

Helper T cells in mice have been divided into two functionally distinct subsets based upon the pattern of cytokines secreted,1,2 with Th type 1 (Th l) cells producing IL-2 and IFN-γ upon activation and predominantly mediating cell-mediated immunity (CMI), and Th type 2 (Th2) cells producing IL-4, IL-5, IL-6, and IL-10 upon activation and primarily mediating humoral immunity by providing T cell help via cytokines for Ig isotype and subclass responses.1–4 For T cell activation, dendritic cells (DC) are the most potent inducers of primary in vivo T cell responses and are the principle in vitro stimulators of naive T cell activation in both mice and humans.5,6 Previous studies showed that DC from different lymphoid tissues could possess similar7 or different8 functions. These studies supported the notion that PP DC induce preferential IgA production (predominantly found at mucosal sites) and SP DC support IgM production by controlling the cytokines produced by T cells in mucosal and systemic tissues, respectively. Recent studies suggest that cytokines present during primary activation of naive T cells play a role in determining the pattern of cytokines produced during subsequent antigenic challenge.9 Perhaps more important to these stimulatory events are the APC or accessory cells (AC) resident in the local tissues that can stimulate initial cytokine secretion in this inductive milieu. In this regard, the present studies have addressed tissue specificity of the AC component of T cell activation using a single type of AC, namely, DC isolated from two different anatomical sites. We asked whether SP DC and PP DC, isolated under identical conditions, induce production of similar levels of T cell-derived cytokines that are involved in regulation of immune responses.

Mucosal Homeostasis: Role of Interleukins, Isotype-specific factors and Contrasuppression in the IgA response

Oral ingestion of antigen elicits immune responses at mucosal sites where humoral immunity is largely due to antibodies of the IgA isotype. This is often accompanied by suppression of systemic responses to the same antigen, a state termed oral tolerance. This IgA response is regulated by interactions between T cell subsets found at IgA inductive tissues, i.e., the gut-associated lymphoreticular tissue (GALT) or Peyers patches (PP). PP T helper (Th) cells support IgA responses, and interleukins 5 (IL-5) and IL-6 can augment secretion of this isotype. Subsets of Th cells may also express Fc receptors for IgA (Fc alpha R) and secrete Fc alpha R as an IgA-binding factor (IBF alpha). Membrane-derived Fc alpha R is a glycoprotein of 38,000 M.W. and this molecule induces selective increases in IgA secreting cells (as determined by the ELISPOT assay) in PP B cell cultures. Fc alpha R+ T cell lines have been shown to secrete IBF alpha as well as IL-5 both of which promote IgA synthesis. Recombinant IL-5 (rIL-5) and rIL-6 induce IgA synthesis mainly by PP B cell blasts, and principally act on surface IgA-positive (sIgA+) B cells for these responses. Another form of mucosal regulation is provided by T contrasuppressor (Tcs) cells, which abrogate oral tolerance when adoptively transferred to mice and restore systemic responsiveness to the antigen sheep erythrocyte (SRBC). Tcs cells from mice systemically primed with SRBC support IgM and IgG subclass responses, while Tcs cells from orally primed mice support IgM, IgG subclass and IgA anti-SRBC responses. These Tcs cells are CD3+, CD4-, 8- and are antigen-specific. These regulatory cells may use the gamma-delta (gamma-delta) form of T cell receptor for antigen recognition.

The regulation of IL-6 secretion from IEC-6 intestinal epithelial cells by cytokines and mucosally important antigens.

Intestinal epithelial cells (IEC) are known to secrete a variety of important cytokines including the inflammatory cytokine interleukin-6 (IL-6).1,2 Along with its role in the inflammatory response, IL-6 has also been shown to act as a co-stimulator for T cell proliferative responses3 and is known to induce Peyer’s patch and appendix B cells to secrete high levels of IgA.4, 5 Therefore, the IEC, through its secretion of IL-6, has the potential to be a very important factor in inflammation and IgA immune responses at the intestinal mucosa.

Divergent T-cell cytokine profiles induced by dendritic cells from different tissues.

Dendritic cells (DC) were originally described by Steinman and Cohn in 1973. 1, 2 Their methods of splenic DC isolation have involved the flotation of murine spleen (SP) cells on dense bovine serum albumin (BSA) gradients and differential glass or plastic adherence of SP cells. Nonadherent Peyer’s patch (PP) DC were isolated in our laboratory by Spalding and coworkers in 1983. 3 These methods employed flotation of PP cells on dense BSA gradients, clustering of DC within the buoyant PP population with periodate-modified SP T cells, dissociation of clusters, and final BSA flotation of enriched PP DC.