Regulation of mucosal IgA responses in vivo: cytokines and adjuvants

Veterinary

immunology and

Veterinary Immunology and Immunopatbology 54(1996) 179-186

ELSEVIER

Regulation of mucosal IgA responses in vivo: cytokines and adjuvants Alan J. Husband aT* , David R. Kramer a, Shisan Bao a, Robyn M. Sutherland a, Ken W. Beagley b aDepurtment c$Veterinury Pathology, The University of’Sydney. Sydney. N.S.W. 2006, Australia b Fuculty ofMedicine. The University ofNewcastle. Newcastle. N.S.W. 2308, Austrulio Accepted 8 July 1996

Abstract The predominance of IgA plasma cells at mucosal sites reflects a combination of the selective localisation and vigorous proliferation after extravasation of IgA plasma cell precursors. Experiments are described here which demonstrate that post-extravasation events leading to IgA precursor cell retention, proliferation and antibody secretion are under cytokine control. This has led to investigation of therapeutic interventions to modify cytokine availability to maximise mucosal vaccination responses and correct IgA deficiencies. Keywords: Mucosal immunity; IgA; Cytokines; Gene therapy; IL-6

1. Introduction

Diseases affecting mucosal surfaces remain as the greatest cause of mortality and morbidity in both man and animals. For this reason, attempts to understand the immune mechanisms operating at mucosal sites, and to develop strategies for improving these responses have received increasing attention. The first line of defence at mucosal surfaces is undoubtedly provided by the secretion of IgA antibodies into the mucus layer overlying the mucosa. The rate of IgA secretion is directly proportional to the number of 1gA plasma cells located in the submucosa since the bulk of IgA antibody in secretions is locally produced. Understanding the factors which determine the successful induction

Abbreviations: AOCC = Anti-ovalbumin-containing cells; HA = Haema,,oolutinin; ID = Intraduodenal; IP = Intraperitoneal; LP = Lamina propria: PP = Peyer’s patch ’ Corresponding

author.

0 165.2427/96/$15.00 Copyright PI1 SO1 65.2427(96)05688-7

6 1996 Elsevier Science

B.V. All rights reserved

of IgA precursor cell recruitment and isotype commitment in mucosal inductive sites and their subsequent localisation in effector sites will enable a rational approach to mucosal vaccine design. It is now accepted that, although there are many factors which influence the induction and dissemination of cells committed to IgA antibody production, they almost certainly work through a common pathway involving cytokine mediators. Thus, the question of how to maximise the mucosal response has focused attention on strategies to upregulate the production of selected cytokines and to improve antigen presentation and processing at these sites. The development of strategies to directly regulate cytokine production coupled with a search for formulations suitable for delivery of response modifiers to mucosal surfaces have become a strong focus for mucosal vaccine research. This paper addresses the role of cytokines in controlling the effector phase of mucosal responses involving the localisation of IgA precursor cells in mucosal effector sites and their subsequent retention and proliferation, and delivery formulations which may be useful in cytokine therapy applications. It is well-documented that the majority of IgA plasma cell precursors arise in Peyer’s patches (PP), after engagement with luminally derived antigen, and then enter the circulation from which they have the potential to migrate to a variety of mucosal subepithelial sites. In addition. a minor population of IgA plasma cells in the intestine arise from CDS ’ B cell precursors (B-l cells) originating in the peritoneal cavity (Solvason et al.. 1991). The density of IgA plasma cells at mucosal sites is a reflection of the combined effects of factors influencing extravasation and factors influencing post-extravasation persistence. Factors influencing extravasation include receptor-ligand interactions at the endothelium (Nakache et al., 1989) allowing precursors of IgA plasma cells to extravasate into mucosal tissues in a selective fashion. However, the major factor determining post-extravasation persistence is the rate of local proliferation. This was demonstrated by adoptively transferring IgA precursor cells from intestinally immunised donor animals into similarly immunised recipients in which the source of endogenous IgA precursor cells was diverted by thoracic duct cannulation. By chasing the adoptively transferred cell inoculum with an intravenous dose of tritiated thymidine. it was possible by autoradiography to detect division of the transferred cells after they had extravasated into the intestinal lamina propria (LP) tissues (Fig. I) (Husband et al.. 1977). Thus, the distribution of IgA plasma cells at mucosal sites is highly dependent on local factors providing proliferative signals to incoming plasma cell precursors. While local antigen undoubtedly contributes to this signal (Husband, 19851, subsequent experiments have indicated that adoptively transferred immune T cells can substitute for antigen to produce apparent local IgA cell retention (Dunkley and Husband, 1991). These findings provoked investigations of the role of T cell cytokines in the effector phase of mucosal IgA responses.

2. Role of cytokines

in IgA production:

in vitro studies

It is apparent from in vitro studies that the T dependency of IgA responses (Kawanishi et al.. 1983) is the reflection of two distinct signals-one involving direct

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contact with the CD4 T cell membranes and one provided by T cell cytokines (Hodgkin et al., 199 1). Mosmann and Coffman ( 1989) described a phenotypic polarisation among CD4+ T cells in respect of their cytokine profiles-those T cells which appeared to promote mainly cell-mediated immunity (Th 1 cells) were shown to produce mainly IL-2 and IFN, while those cells promoting antibody responses (Th2 cells) produced mainly IL-4, IL-5, IL-6 and IL- 10. In this regard, T cells isolated from mucosal lymphoid tissue are heavily geared towards Th2 responses (Xu-Amano et al., 1994). This observation is consistent with in vitro data demonstrating that Th2 cytokines promote IgA expression by surface IgA+ B cells derived from PP and encourage proliferation of IgA committed B cells in the absence of T cell contact (Beagley et al., 1988; Beagley et al., 1989: Kunimoto et al., 1989; Defiance et al., 1992; Sonoda et al., 1992). It is therefore apparent that the expression of these cytokines in the LP microenvironment in vivo may be the key factor determining the number of IgA plasma cells and their level of IgA expression and secretion in these sites.

3. Role of cytokines

in IgA production:

in vivo studies

Experiments in this laboratory have focused more recently on the role of Th2 cytokines in IgA expression in the intestine in vivo. Since in vitro studies suggest that there is a predominance of cells expressing Th2 cytokines in mucosal sites, we have used in situ hybridisation techniques to detect cytokine mRNA to determine the distribution of cytokine expression in the normal mouse intestine. Cells expressing mRNA for IL-4, IL-5 and IL-6 (cytokines which promote induction and expression of IgA in vitro) are distributed throughout the LP, closely mapping the distribution of IgA plasma cells (see Fig. 1) but cells expressing mRNA for IFN, a cytokine which downregulates IgA expression, are only distributed in the basal regions of the LP adjacent to the muscularis, and remote from the microenvironment where IgA production occurs (Baa et al., 1993). The recent availability of mice with targeted disruption of genes coding for various cytokines has provided the opportunity to study more closely the role of cytokines in vivo. IL-4 gene knockout mice (IL-4-/-J have deficient IgGl and IgE responses and fail to develop Th2 cytokine secreting cells following nematode infection, but neither levels of serum or intestinal lavage IgA nor numbers of LP IgA plasma cells are affected (Kopf et al., 1993). However, these mice fail to mount an intestinal IgA response after oral immunization with ovalbumin or KLH, despite a normal IgA response to co-administered cholera toxin (Vajdy et al., 1995). These data suggest that IgA responses to protein antigens within the PP may be dependent on IL-4 expression and the consequent cascade of Th2 cytokine responses. IL-6 appears to be much more important for maintenance of an IgA effector response in vivo since IL-6-/mice have a 60% reduction in total IgA plasma cells in the intestinal LP (Ramsay et al., 1994). Investigation of the nature of the residual IgA plasma cells in IL-6 deficient mice revealed that these were enriched for CD5+/IgA+ cells. presumably derived from peritoneal cavity B-I precursors which have been shown to be IL-6 independent (Beagley et al., 1995). The deficiency in conventional PP-derived

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IgA cells in these mice is reflected in an inability to develop antigen-specific IgA-containing cells in the intestine following local challenge with ovalbumin or to develop haemagglutinin (HAlspecific IgA secreting cells in lung following aerosol immunization with a vaccinia virus vector expressing influenza HA (Ramsay et al., 1994). However, overexpression of IL-6 in transgenic mice (Suematsu et al., 1989) or in mice reconstituted with haematopoietic stem cells containing a retroviral insertion of IL-6 (Brandt et al., 1990) resulted in a two- to three-fold increase in serum IgA. 4. Cytokine therapy The key role which cytokines play in regulation of IgA responses raises the prospect of administration of cytokines as a form of adjuvant. In this regard, Pockley and Montgomery (1991) demonstrated increased IgA expression in lacrimal glands after local injection of IL-5 and IL-6 More recent data (Else et al., 1994) indicate that whereas depletion of IFN resulted in expulsion of Trichuris muris from the intestine of a strain of mice normally unable to expel the parasite, the administration of IL-4 facilitated expulsion and enabled established adult worms to be expelled when administered late in infection. However, administration in vivo of IFN- Cl (Urban et al., 1993) or IL-l 2 (Morris et al., 1994) inhibited intestinal worm expulsion through downregulation of IL-4. In our own laboratory we have investigated the potential for exogenous IL-6 to boost intestinal responses to soluble antigen. Rats were primed IP with ovalbumin in FCA, a procedure which does not produce an intestinal response but primes the intestine for a vigorous IgA-specific anti-ovalbumin-containing cell (AOCC) response in the intestine after subsequent intraduodenal (ID) challenge (Husband and Gowans, 1978). Double Thiry-Vella loops were prepared 14 days after priming and an ID challenge dose of antigen administered. Preliminary data (Table 1) indicate that administration of ovalbumin with recombinant IL-6 to the proximal loop produced an enhanced AOCC response compared with the distal loop receiving ovalbumin alone. An enhanced effect was observed if antigen + cytokine was administered on two occasions (days 16 and 17). By providing additional precursor cells, by adoptive transfer of thoracic duct lymphocytes collected from IP/ID immunised donors, the total AOCC response in each loop was further enhanced but the relative improvement in response in the cytokine treated loop was similar.

Fig. I. (a) The appearance of IgA plasma cells in the intestine of a normal mouse. (b) Radiolabelted anti-ovalbumin-containing cell (arrowed) in the intestine of a rat in which adoptive transfer of IgA precursor cells, derived from the thoracic duct of an immune donor, was chased by an i.v. pulse of tritiated thymidine, timed to occur after the precursors had extravasated into the intestinal lamina propria. This demonstrates that post-extravasation proliferation occurs and is a factor determining the locahsation and magnitude of intestinal antibody responses. Note also dense labclling of crypt epithelial cells, consistent with these cells undergoing rapid division. (Autoradiographic grains appear as bright dots due to dark field illumination.) (c) The distribution of mRNA for IL-6 in the intestine of a normal mouse detected by in situ hybridisation using a radioactively labelled antisense oligonucleotide probe. Note that the distribution of IL-6-producing cells is similar to that of IgA plasma cells shown in (a) and is a potential factor in providing proliferative signals for incoming IgA plasma cell precursors. (Autoradiographic grains appear as black dots.)

A.J. Hushmui

184

Table I Enhancement Exp. b

of anti-ovalbumin

2 3

containing

Immunology

und Immunoputhol~~~y

cells (AOCC)

m Thiry-Vella

Treatment day 16

Treatment day 17

TDL’ day 17

Prox. Dist. Prox. Disk Prox.

No No Ova + IL-6 Ova Ova + IL-6

Ova+ IL-6 ’ ova f Ova + IL-6 Ova Ova + IL-h

Dist.

Ova

Ova

Loop

“0.

I

et 01. / Vetrrirwy

54 f1996)

179-186

loops ’ by exogenous

IL-6

AOCC d day 19

Ratio prox:dist

Total AOCC

IgA AOCC

Total AOCC

kA AOCC

No

44

ND ND

1.26

ND

No

35 II9

57

I.35

1.32

Ye.\

XX 260

43 187

1.27

I .28

205

146

AAdult PVG rats were primed on Day 0 by intraperitoneal administration of 0. I mg of ovalbumin emulsified in complete Freund’s adjuvant. Thiry-Vella loops were constructed on day 14, at which time the animals received an intraduodenal boost of 0.5 mg ovalbumin in PBS. b Each experiment consists of data from one animal, except Experiment 3 in which the mean data from two animals are presented. ’ Adoptive transfer of 5 X IO’ thoracic duct lymphocytes from donors immunised as described above. d Data expressed as cells per linear centimetre of intestine in the plane of the section, as previously described (Husband and Gowans, 1978). ’ Intra-lumenal administration into Thiry-Vella loop of 0.5 mg ovalbumin in PBS+ I X IO’ U recombinant murine IL-6. f Intra-lumenal administration mto Thiry-Vella loop of 03 mg ovalbumin in PBS.

Increasing appropriate cytokine expression at mucosal sites is an alternative approach to selectively enhance IgA responses. A range of genetically engineered replicating vectors carrying cytokine gene insertions are now available for this purpose. For instance, using a vaccinia vector, we have shown that IL-6 gene therapy can restore the deficient IgA responses in IL-6 gene knockout mice. Whereas IL-6 knockout mice are unable to produce an IgA-specific anti-HA response when given a respiratory tract infection with recombinant vaccinia engineered to express the HA gene, the HA-specific IgA response was restored if they were infected with recombinant virus co-expressing HA with IL-6 (Ramsay et al., 1994). Even normal mice infected with recombinant vaccinia expressing a variety of Th2 cytokines display enhanced IgA responses in tissues adjacent to the focus of infection (Ramshaw et al., 1992). These studies demonstrate the potential for cytokine gene therapy. not only to reconstitute defective responses, but to enhance normal responses. Other viral vectors such as adenovirus (Xing et al.. 1994) and fowl pox virus (Leong et al.. 1994) have shown promise as mucosal cytokine gene delivery vectors, the latter being of particular interest because of its low risk of pathogenicity in mammalian species. Naked DNA gene transfection technologies may provide a means of overcoming problems associated with replicating vectors-the risk of reversion to virulence, adverse reactions, especially in individuals who are immunocompromised, and the vector-specific response stimulated in the immunised host, which limits the subsequent usefulness of the same vector. This approach has been shown to create a transient transgenic state in mice injected with plasmid DNA containing a selected gene driven by a strong eukaryotic promoter (Wolff et al., 1990) and has been used as an effective method of vaccination against influenza virus by injecting mice intramuscularly with DNA encoding influenza

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nucleoprotein linked to the human cytomegalovirus promoter (Ulmer et al., 1993). It has also been demonstrated that skin and mucosal routes of inoculation can be used successfully to achieve host expression of plasmid vectored genes (Webster et al., 1994). It appears that the less efficient transfection rates which occur in these tissues may be offset by highly efficient antigen presentation and recognition. There is considerable potential for the development of naked DNA techniques to upregulate local cytokine expression using constructs consisting of genes encoding for cytokines, or other response modifiers, administered by mucosal routes. For instance, it might be desirable to employ primary immunisation targeted to PP with a construct encoding for a relevant antigen together with IL-4 and/or TGF genes to enhance induction of IgA responses. Then, in view of the key role which IL-6 plays in promoting localisation and proliferation of IgA plasma cells in mucosal effector sites, administration of IL-6 gene constructs could boost and maintain local antibody expression.

5. Future developments The potential for manipulating cytokines is now established and there is sufficient data available on both cytokine effects and intervention strategies to enable practical outcomes to be envisaged. There are a number of key areas where cytokine therapies may contribute. Alteration in cytokine profile have been used to maxim& mucosal vaccination responses (Pockley and Montgomery, 1991; Nash et al., 1993) and to correct acquired or congenital immunodeficiency disorders (Briere et al., 1994; Eisenstein et al., 1994). However, if either soluble cytokine or plasmid vectors are to be used to achieve cytokine effects at mucosal sites, effective delivery formulations will be required. Liposomes have proved to be effective in delivering cytokines to mucosal induction and effector sites (Abraham and Shah, 1992; Duits et al., 1993) and polymer microcapsules act in a similar manner to liposomes but are more effective in protecting antigen from gastric degradation following oral delivery allowing selective absorption by M cells in the PP (Greenway et al., 1995; Muir et al., 1994). The ability of DL-lactide-co-glycolide microcapsules to provide release characteristics ranging from hours to months raises the prospect of formulations which enable the cytokine profile to be manipulated differentially over time (e.g. IL-4 release at first followed later by IL-6 release). The further development of these strategies will open new opportunities for mucosal vaccination and immunomodulation.

Acknowledgements

This study was supported by the National Health and Medical Research Council of Australia.

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