Michael Berman

Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production

To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.

T-cell properties determine disease site, clinical presentation, and cellular pathology of experimental autoimmune encephalomyelitis.

Two distinct clinical phenotypes of experimental autoimmune encephalomyelitis are observed in BALB interferon-gamma knockout mice immunized with encephalitogenic peptides of myelin basic protein. Conventional disease, characterized by ascending weakness and paralysis, occurs with greater frequency after immunizing with a peptide comprising residues 59 to 76. Axial-rotatory disease, characterized by uncontrolled axial rotation, occurs with greater frequency in mice immunized with a peptide corresponding to exon 2 of the full length 21.5-kd protein. The two clinical phenotypes are histologically distinguishable. Conventional disease is characterized by inflammation and demyelination primarily in spinal cord, whereas axial-rotatory disease involves inflammation and demyelination of lateral medullary areas of brain. Both types have infiltrates in which neutrophils are a predominating component. By isolating T cells and transferring disease to naive recipients, we show here that the type of disease is determined entirely by the inducing T cell. Furthermore, studies using CXCR2 knockout recipients, unable to recruit neutrophils to inflammatory sites, show that although neutrophils are critical for some of these T cells to effect disease, there are also interferon-gamma-deficient T cells that induce disease in the absence of both interferon-gamma and neutrophils. These results highlight the multiplicity of T-cell-initiated effector pathways available for inflammation and demyelination.

Latent collagenase in the ulcerating rabbit cornea

“Latent” collagenase† has been activated by brief trypsin treatment of crude culture media protein from both ulcerating and non-ulcerating (normal) rabbit corneas. Collagenase activity generated in this way is able to cleave re-constituted collagen in fibrils and cleaves soluble collagen into 34- and 14-length fragments in the manner of the vertebrate collagenases. Collagenase activity has been generated also by trypsin treatment of molecular sieve fractions containing protein only slightly larger in size than the manifest collagenase of approximately 40 000 mol weight. Both latent and manifest collagenase treated with trypsin elute with an apparent molecular weight of 23 000 and are capable of being bound by the serum antiprotease, α2-macroglobulin. The nature of the latent collagenase is not known. It may be a complex of collagenase with an inhibitor, or it may be a zymogen.

Corneal ulceration and the serum antiproteases. II. Complexes of corneal collagenases of α-macroglobulins

Previous work has demonstrated that whole rabbit or human serum and the human α2-macroglobulin (Hα2-m) inhibit corneal collagenases. In the present study, the ability of rabbit and human serum to cause rabbit corneal collagenase, mol. wt 45 000, to elute in high molecular weight fractions from sieving columns is taken as presumptive evidence for the formation of collagenase-serum protein complexes. The recovery of increased collagenase activity by thiocyanate treatment of serum effluent fractions containing the human α2-macroglobulin indicates that it is the α2-m in human serum that complexes rabbit corneal collagenase. This conclusion is supported by the demonstration that purified Hα2-m transports the rabbit corneal collagenase through a molecular sieve. Moreover, the chromatography of day one culture media from ulcerating rabbit corneas has demonstrated the presence of a significant amount of the total collagenase activity in the high molecular weight (850 000 – 1 000 000) fractions in which rabbit α-macroglobulin (Rα1-m) was also demonstrated. These observations support the hypothesis that the α-macroglobulins play an important role in the regulation of corneal collagenase activity. Crossed-gel immunoelectrophoretic methods have shown that rabbit and human corneal collagenase preparations perturb the patterns of their respective α-macroglobulins. The perturbed patterns are taken as evidence for the formation of collagenase-α-macroglobulin complexes. The application of crossed-gel methods to the tears of human ulcer patients shows the utility of such methods for examining the status of α2-m in tears.

Regulation of collagenase activity in the ulcerating cornea by cyclic-AMP

Abstract Dibutyryl cyclic-AMP (DBcAMP) and theophylline partially inhibit the appearance of collagenase activity when added to cultures of ulcerating rabbit corneas. Drug treatment with DBcAMP also inhibits degradation of collagen in the explanted corneas as measured by hydroxyproline in the media. The hydrolysis product of DBcAMP, 5′-adenosine monophosphate (5′ AMP) is itself very effective in suppressing collagenase activity and the degradation of explant collagen. The mechanism(s) of the drug effects is not yet known but it is possible that the drugs affect the synthesis and/or the secretion of collagenase. The results suggest that “first messengers” exist which can prevent the secretion of corneal collagenase by raising endogenous cAMP in corneal cells through stimulation of the adenyl cyclase system. Eventual successful treatment of corneal ulceration might well require pharmacologic intervention at multiple levels: the biosynthesis, secretion, activation and activity of collagenase.

Characterization of human corneal collagenase

Collagenolytic activities have been detected in the culture media from an eye bank cornea and in media from corneal tissues biopsied at the time of surgery. The eye bank cornea was not pathological but was not used for corneal grafting because of the donor age (77). The corneal biopsies were from keratoplasty patients with no history of corneal ulceration. Enzymes from both sources cleave tropocollagen at the same site as do tadpole-tail collagenase and rabbit corneal collagenases, and, because of the restricted cleavage, belong to the class of enzymes known as “tissue collagenases”. The human corneal collagenase activities also degrade reconstituted collagen fibrils. The collagenase activity(ies) from human keratoplasty tissues is inhibited by Calcium-EDTA and N-acetyl-l-cysteine, both of which have been found previously to prevent ulceration in the alkali-burned rabbit cornea and to inhibit collagenases produced by the ulcerating rabbit cornea. The serum antiprotease, α2-Macroglobulin, has also been found to inhibit the collagenase(s) from keratoplasty tissues, an observation which supports the ophthalmologists sometime contention that serum inhibits corneal ulceration. It is hoped that the human corneal collagenase(s) produced by the culture of keratoplasty tissues and of eye bank corneas judged unfit for corneal grafting will facilitate the discovery of effective inhibitors of corneal ulceration.

5′ Adenosine monophosphate prevents collagen degradation in culture but does not prevent corneal ulceration

Abstract Alkali-burned rabbit corneas were treated topically with 5′ adenosine monophosphate (5′ AMP) in an attempt to prevent corneal ulceration. Drug treatment did not reduce the frequency or the severity of ulceration compared to treatment with an osmotically-matched saline control. Ulcerating corneas were cultured subsequently in control medium or in medium containing 5′ AMP. Corneas that had received saline in vivo produced high levels of collagenase activity and showed extensive explant degradation when cultured in control medium. When cultured in the presence of 5′ AMP, however, such corneas produced barely-detectable levels of collagenase, and explants were not apparently degraded. Corneas that had received 5′ AMP in vivo also produced high levels of collagenase activity and showed extensive explant degradation when cultured in control medium. When cultured in medium containing 5′ AMP, however, such corneas showed minimal degradation as evidenced by their appearance and by the very low hydroxyproline levels in the media. Significantly, media harvested from such cultures contained high levels of collagenase activity.