Robert J. Hohman

Substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide increase in nasal secretions after allergen challenge in atopic patients

BACKGROUND There is suggestive evidence that neuropeptides participate in allergic reactions. Substance P (SP) and calcitonin gene-related peptide (CGRP) are released by sensory nerves, whereas vasoactive intestinal peptide (VIP) is released mainly by parasympathetic nerves. Both sets of nerves are thought to be stimulated by allergic inflammation. The aim of this study was to assess nasal secretions to determine whether SP, CGRP, and VIP were increased after allergen challenge. METHODS Eight patients with allergic rhinitis were challenged nasally with 1 mg histamine or increasing doses of allergen. Nasal lavages were collected into a cocktail of protease inhibitors in order to restrict neuropeptide degradation. Radioimmunoassay for SP, CGRP, and VIP were performed on each sample. RESULTS All patients had immediate clinical reactions to both histamine and allergen challenges, and seven patients experienced a later allergic reaction. After histamine challenge, SP and CGRP did not increase significantly above baseline in the nasal lavages, whereas VIP did (p < 0.02). In contrast, SP, CGRP, and VIP all significantly increased immediately after allergen challenge and returned to baseline within 2 hours. At the clinical peak of the late allergic reaction, SP, but not CGRP or VIP, was increased slightly but significantly (p < 0.01). CONCLUSIONS Thus SP, CGRP, and VIP are found in nasal secretions after allergen challenge, which confirms that neuropeptides are released in human beings during allergic reactions. The selective stimulation of VIP secretion by histamine challenge suggests that histamine-induced cholinergic reflexes induce the release of VIP. These data support the suggestion that neuropeptides may be partly responsible for some of the nasal symptoms of allergy.

Aminopeptidase activity in human nasal mucosa

Abstract Background: Aminopeptidases activate bradykinin and degrade many inflammatory peptides. Objective: The objective of this study was to identify the types of aminopeptidase activities in human nasal mucosa. Methods: Human nasal mucosa was homogenized (n = 12), and cytoplasmic (S2) and membrane-rich (P2) fractions were obtained. Several aminopeptidase (Ap) activities were defined by (1) substrate specificity with leucine-enkephalin (leu-Ap) and alanine-nitroanilide (ala-Ap), (2) inhibitor studies with puromycin and bestatin, (3) enzyme activity histochemistry (zymography), (4) immunohistochemistry, and (5) gel electrophoresis. Human volunteers had methacholine, histamine, and allergen nasal provocations to determine the mechanisms controlling nasal aminopeptidase secretion in vivo. Results: P2 was the largest reservoir of puromycin-resistant aminopeptidase activity (630 pmol leu-enk/min/mg protein). S2 contained 32 pmol leu-enk/min/mg activity, with 80% representing puromycin-resistant activity and 20% puromycin-sensitive aminopeptidase (PS-Ap). Ala-Ap was detected in both P2 and S2 fractions and was localized by zymography to epithelial and gland cells. Anti–rat brain–soluble PS-Ap IgG detected immunoreactive material in epithelium, glands, and endothelium. In nasal provocation studies, leu-AP correlated with glandular exocytosis but not vascular leak. Conclusions: The predominant aminopeptidase in human nasal epithelial and submucosal gland cells was membrane-bound puromycin-resistant aminopeptidase. A novel soluble puromycin-resistant aminopeptidase and lower amounts of soluble PS-Ap were also detected. (J Allergy Clin Immunol 1998;102:741-50.)

Use of AMP specific antibodies to differentiate between adenylylated and unadenylylated E. coli glutamine synthetase.

Abstract Anti-AMP specific antibodies were purified by affinity chromatography of serum from sheep immunized with adenylylated bovine serum albumin. Results of immunotitration experiments and light scattering measurements show that these antibodies can be used to separate adenylylated from unadenylylated forms of E. coli glutamine and to detect variations in protein configurations elicited by partial adenylylation of the enzyme or by allosteric interactions with divalent cations. These results suggest that the reaction of anti-AMP antibodies with variously adenylylated forms of glutamine synthetase can be used to investigate the dependence of immunoprecipitability on the density, absolute numbers, and possibly, the spatial distribution of multiple identical antigenic sites on a given macromolecule.

Relationship between epitope density and immunoprecipitation of multivalent antigens by bivalent antibody: Immunoprecipitation of adenylylated glutamine synthetase by anti-AMP antibodies

Abstract Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS 12 − ) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS 6 − ) are only partially precipitated by the antibodies ( R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980 , Proc. Nat. Acad. Sci. USA 77 , 7410–7414). By means of 125 I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS 6 − or GS 12 − contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS 12 − , whereas GS 6 − binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS 6 − , the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS 12 − -Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS 6 − preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS 6 − preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS − 12 by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS 6 − with anti-AMP antibodies is greater at 30 °C than at 4 °C.