A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin alpha-dependent fashion.

Lymphotoxin (LT)alpha is expressed by activated T cells, especially CD4(+) T helper type 1 cells, and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LTalpha-deficient (LTalpha-/-) mice. LTalpha-/- mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very low levels of IgG after immunization with sheep red blood cells. We show here that LTalpha-expressing B cells are essential for the recovery of primary, secondary, and memory humoral immune responses in LTalpha-/- mice. It is not necessary for T cells to express LTalpha to support these immune functions. Importantly, LTalpha-expressing B cells alone are essential and sufficient for the formation of FDC clusters. Once these clusters are formed by LTalpha-expressing B cells, then LTalpha-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.

Lymphotoxin-alpha (LTalpha) supports development of splenic follicular structure that is required for IgG responses.

LTalpha-deficient (LTalpha-/-) mice show altered splenic microarchitecture. This includes loss of normal B cell-T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTalpha-/- mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTalpha expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTalpha-/- mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTalpha-/- mice, follicle structure and a strong IgG response were retained. These data indicate that LTalpha-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTalpha-/- mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTalpha could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTalpha-/- mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTalpha-/- BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTalpha supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.

Distinct roles of lymphotoxin alpha and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells.

In mice deficient in either lymphotoxin alpha (LT-alpha) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-alpha and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-alpha-deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-alpha-expressing cells required to establish organized FDC are derived from BM. The role of LT-alpha in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)-deficient BM cells into LT-alpha-deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-alpha-deficient recipient. Thus, expression of LT-alpha in the BM-derived cells, but not in the non-BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I-deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non-BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I-deficient BM was able to restore FDC organization and GC formation in LT-alpha-deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-alpha-expressing BM-derived cells and by TNFR-I-expressing non-BM-derived cells.

Essential role of lymph nodes in contact hypersensitivity revealed in lymphotoxin-alpha-deficient mice.

Lymph nodes (LNs) are important sentinal organs, populated by circulating lymphocytes and antigen-bearing cells exiting the tissue beds. Although cellular and humoral immune responses are induced in LNs by antigenic challenge, it is not known if LNs are essential for acquired immunity. We examined immune responses in mice that lack LNs due to genetic deletion of lymphotoxin ligands or in utero blockade of membrane lymphotoxin. We report that LNs are absolutely required for generating contact hypersensitivity, a T cell-dependent cellular immune response induced by epicutaneous hapten. We show that the homing of epidermal Langerhans cells in response to hapten application is specifically directed to LNs, providing a cellular basis for this unique LN function. In contrast, the spleen cannot mediate contact hypersensitivity because antigen-bearing epidermal Langerhans cells do not access splenic white pulp. Finally, we formally demonstrate that LNs provide a unique environment essential for generating this acquired immune response by reversing the LN defect in lymphotoxin-alpha(-/)- mice, thereby restoring the capacity for contact hypersensitivity.

Mice deficient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone.

Mice rendered deficient in IL-1 beta by gene targeting in embryonic stem cells develop and grow normally in a protected laboratory environment. Endotoxin-stimulated peritoneal macrophages from IL-1beta-deficient mice showed normal synthesis and cellular release of IL-1alpha after treatment with 5 mM ATP demonstrating that IL-1beta is not necessary for expression and release of the IL-1alpha isoform. Mice deficient in IL-1beta showed unaltered sensitivity to endotoxic shock, with or without pretreatment with D-galactosamine. In contrast, IL-1beta-deficient mice showed defective contact hypersensitivity responses to topically applied trinitrochlorobenzene (TNCB). This defect could be overcome either by application of very high doses of sensitizing antigen, or by local intradermal injection of recombinant IL-1beta immediately before antigen application. These data demonstrate an essential role for IL-1beta in contact hypersensitivity and suggest that IL-1beta acts early during the sensitization phase of response. They suggest an important role for IL-1beta in initiation of the host of response at the epidermal barrier.

The effects of thrombin on phytohemagglutinin receptor sites in human platelets.

We have previously demonstrated that lentil phytohemagglutinin (lentil-PHA) binds to human platelet membranes without causing either aggregation or the release reaction. When platelets are treated with thrombin, there is an increase in lentil-PHA binding suggesting the appearance of new receptor sites on the cell surface. We prepared a lentil-PHA-ferritin conjugate using affinity chromatography which was used to saturate cell surface receptor sites. Studies using this conjugate suggest that thrombin causes a complex change in the platelet surface involving a decrease in the number of lentil-PHA receptor sites on the external platelet surface with a marked increase in sites within the center of the canalicular system. These increased sites may result from fusion of granule membranes with the canalicular membranes during the secretion process. There is no obvious relationship between lentil-PHA receptor sites and intramembranous particles.

The role of CCR7 in TH1 and TH2 cell localization and delivery of B cell help in vivo.

Subsets of murine CD4+ T cells localize to different areas of the spleen after adoptive transfer. Naïve and T helper 1 (TH1) cells, which express the chemokine receptor CCR7, are home to the periarteriolar lymphoid sheath, whereas activated TH2 cells, which lack CCR7, form rings at the periphery of the T cell zones near B cell follicles. Retroviral transduction of TH2 cells with CCR7 forces them to localize in a TH1-like pattern and inhibits their participation in B cell help in vivo but not in vitro. Thus, differential expression of chemokine receptors results in unique cellular migration patterns that are important for effective immune responses.

Germline organization of the murine T cell receptor beta-chain genes.

The complete germline organization of the beta-chain genes of the murine T cell receptor was elucidated in order to obtain the structural basis for understanding the mechanisms of somatic DNA rearrangements. Twenty of the 22 known variable (V beta) genes are clustered within 250 kilobases of DNA 5' to the constant region (C beta) genes. These V beta genes share the same transcriptional orientation as the diversity (D beta), joining (J beta), and C beta genes, which implies that chromosomal deletion is the mechanism for most V beta to D beta-J beta rearrangements. Within this V beta cluster, the distance between the most proximal V beta gene and the D beta-J beta-C beta cluster is 320 kilobases, as determined by field-inversion gel electrophoresis. The large distance between V beta and D beta, relative to that between D beta and J beta, may have significant implications for the ordered rearrangement of the T cell receptor beta-chain genes.

Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.

In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.

Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation.

We have studied the actions of helper T lymphocyte-1 and -2 (Th1 and Th2) cells in an acute model of eosinophilic airway inflammation by infusing chicken ovalbumin-specific (OVA-specific) Th1 cells, Th2 cells, or both into unsensitized mice and challenging the mice with an OVA aerosol. OVA challenge after infusion of Th1 cells alone resulted in airway inflammation with lymphocytes and monocytes. Challenge after the infusion of Th2 cells alone resulted in minimal inflammation. In contrast, when Th1 and Th2 cells were transferred together, they cooperated to promote a robust eosinophil-predominant inflammatory response. Th1 cells alone were readily recruited to the airways after challenge, but in the absence of Th1 cells, Th2 cells did not accumulate in the airways. When transferred together, both Th1 and Th2 cells, as well as endogenous eosinophils, were effectively recruited. This recruitment was correlated with increased VCAM-1 expression in the medium- and large-sized vessels of the lung and could be inhibited by treating the mice with neutralizing antibodies to TNF-alpha or VCAM-1. These data indicate that Th2 cells require signals in addition to antigen for their effective recruitment to the airways. Th1 cells can provide these signals.

Expression of complement proteins C2 and factor B in transfected L cells.

Factor B and C2 are structurally and functionally similar complement proteins encoded by genes that are closely linked within the class III region of the major histocompatibility complex (MHC). In this study, restriction endonuclease digestion of cosmid DNAs isolated from an H-2d murine genomic library indicated that the chromosomal organization of these two genes was similar in mouse to that in man. To further characterize their expression, cosmid DNAs encoding human and murine factor B and C2 were introduced into mouse L cells by DNA-mediated gene transfer. Factor B expression was demonstrated in cells transfected with either the human or the murine gene, but not in cells transfected with a control plasmid. Synthesis and secretion of factor B by L cells transfected with the human and murine cosmids was similar to that of human and murine cells in primary culture. An interspecies variation between human and murine factor B was identified and reproduced with extraordinary fidelity by the mouse fibroblast. In contrast, C2 RNA and protein were expressed by L cells alone and by L cells transfected with a control plasmid, as well as by L cells transfected with cosmids encoding human and murine complement genes. Expression of the transferred human C2 gene was demonstrated by the presence of a new distinct C2 RNA transcript and secretion of biologically active human C2. These results demonstrate the similarity of organization of the murine and human class III MHC regions. Expression of the two closely linked gene products, C2 and factor B, after DNA-mediated gene transfer provides a system for further analysis of regulation in both normal and deficient states.

Cytokine regulation of secondary lymphoid organ development.

Lymphotoxin and tumor necrosis factor provide essential signals for the formation of secondary lymphoid tissue structures. Lymphotoxin in its membrane form (LT alpha 1 beta 2 heterotrimer) is required for the development of lymph nodes and Peyer's patches and supports the development of normal spleen structure. In the spleen, lymphotoxin acts during embryonic development to support the formation of distinct B and T cell zones. Lymphotoxin also acts in a tonic fashion-supporting the formation and maintenance of the follicular dendritic cell network and of primary B cell follicle structure. The cells that deliver the tonic lymphotoxin signal supporting follicular dendritic cell structure are B cells; thus, B cells participate fundamentally in the development of the lymphoid tissue structure in which they subsequently mature.

A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

Interleukin-1beta promotes repair of the CNS.

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine associated with the pathophysiology of demyelinating disorders such as multiple sclerosis and viral infections of the CNS. However, we demonstrate here that IL-1beta appears to promote remyelination in the adult CNS. In IL-1beta(-/-) mice, acute demyelination progressed similarly to wild-type mice and showed parallel mature oligodendrocyte depletion, microglia-macrophage accumulation, and the appearance of oligodendrocyte precursors. In contrast, IL-1beta(-/-) mice failed to remyelinate properly, and this appeared to correlate with a lack of insulin-like growth factor-1 (IGF-1) production by microglia-macrophages and astrocytes and to a profound delay of precursors to differentiate into mature oligodendrocytes. Thus, IL-1beta may be crucial to the repair of the CNS, presumably through the induction of astrocyte and microglia-macrophage-derived IGF-1.

Factor H.

While the mouse and human H proteins are structurally and functionally similar, they differ in their genetics. Whereas there is no evidence in humans for more than one gene; in mice the H locus is complex. Based on cDNA sequence and hybridization analysis of genomic cosmid clones, there are at least three distinct genes, all highly related to one another. The consensus repeating unit that comprises this molecule has obviously been duplicated numerous times, since it is present in many other molecules. Thus, it is not surprising to discover that there are several genes related to H in the mouse. A similar case has been described for two other members of this family. In humans, CR1 cDNA hybridizes to two distinct genomic clusters in the CR1 locus (Wong et al. 1989), and in mice, mCRY hybridizes to two regions in the genome, one on chromosome 1 and another on chromosome 8 (Aegerter-Shaw et al. 1987). It will be of interest to see if any other members of this family display as complex a genetic locus as murine H.

Different isozymes of mouse 11 beta-hydroxylase produce mineralocorticoids and glucocorticoids.

We have isolated and characterized two isozymes of mouse steroid 11 beta-hydroxylase (11 beta-OHase), designated 11 beta-OHase and aldosterone synthase (AS). Physical mapping of overlapping cosmid and phage isolates defined two genes (designated Cyp11b-1 and Cyp11b-2 in the standard nomenclature for cytochrome P450 genes) that are oriented in the same direction and separated by approximately 8 kilobase pairs of DNA. The two genes are highly homologous in their coding regions, with 84% nucleotide identity and 86% predicted amino acid identity. In regions where the sequences of the rat 11 beta-OHase and AS genes diverged most widely, the mouse sequences also differed significantly, thereby identifying putative mouse 11 beta-OHase and AS genes. Both genes were mapped to chromosome 15 by analyzing restriction fragment length variations in a panel of DNA samples from an interspecific cross. To determine the functional properties of the 11 beta-OHase and AS proteins, we transfected COS-7 cells with plasmids that expressed the proteins encoded by the 11 beta-OHase and AS genes. When expressed in transfected COS-7 cells, the 11 beta-OHase protein converted deoxycorticosterone to corticosterone but did not produce aldosterone. Consistent with its postulated role in mineralocorticoid biosynthesis, the product of the AS gene efficiently synthesized aldosterone. We next studied the expression of these two isozymes in Y1 adrenocortical tumor cells and in the intact mouse adrenal gland. Although Y1 cells otherwise resemble zona fasciculata cells and express the 11 beta-OHase gene at high levels, transcripts encoded by the AS gene were detected at levels approximately 10-fold lower than the 11 beta-OHase transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage apoptosis in the absence of active interleukin-1 beta-converting enzyme.

The interleukin-1 beta converting enzyme (ICE) is the cysteine proteinase responsible for cleaving the 31-kDa interleukin-1 beta (IL-1 beta) precursor to its active 17-kDa form. In lipopolysaccharide-stimulated cultured macrophages, induction of apoptosis but not necrosis effectively induces conversion of the IL-1 beta precursor to its mature form and results in the concomitant release of the mature cytokine from the cell. To determine whether ICE activity is required for macrophage apoptosis, we have exposed macrophages either to 5 mM ATP or to alloreactive cytolytic T lymphocytes (CTL) in the absence and presence of the ICE inhibitor peptide YVAD-chloromethylketone (YVAD-emk). Activated cells treated with YVAD-emk and ATP or CTL showed no mature IL-1 beta in either the cell lysates or the culture supernatants, indicating effective inhibition of ICE activity; however, the YVAD-treated macrophages showed no detectable change in 51Cr release or nuclear fragmentation, indicating failure to inhibit apoptotic cell death. Thus, in these cells, YVAD-emk uncouples IL-1 beta processing and apoptosis.

IL-1ß in eosinophil-mediated small intestinal homeostasis and IgA production.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1ß (IL-1ß), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-ß, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-ß (transforming growth factor ß). GI eosinophils expressed a relatively high level of IL-1ß, and IL-1ß-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1ß in the small intestine.

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.